WO1998014585A1

WO1998014585A1 - Nucleocapsid gene of seoul virus r22, recombinant plasmid, transformed e. coli and diagnostic agent and vaccine for haemorrhagic fever with renal syndrome

Info

Publication number: WO1998014585A1
Application number: PCT/KR1997/000184
Authority: WO
Inventors: Hyun Su Kim; Wang Don Yoo; Kap Soo Noh; Soo Ok Kim; Sang Bum Moon; Jong Wan Kim; Kuen Taik Chung; Young Chul Shin
Original assignee: Cheil Jedang Corporation
Priority date: 1996-10-04
Filing date: 1997-10-02
Publication date: 1998-04-09
Also published as: AU4473497A

Abstract

The present invention relates to a nucleocapsid gene of Seoul virus R22 strain, a recombinant expression vector for the said gene, a transformed microorganism with the said recombinant plasmid and a nucleocapsid protein expressed by the said transformant, as well as a novel pharmaceutical composition for diagnosis or prevention of haemorrhagic fever with renal syndrome caused by viruses in the Hantavirus genus containing the said nucleocapsid protein.

Description

NUCLEOCAPSID GENE OF SEOUL VIRUS R22, RECOMBINANT PLASMID, TRANSFORMED E. COLI AND DIAGNOSTIC AGENT AND VACCINE FOR HAEMORRHAGIC FEVER WITH RENAL SYNDROME

BACKGROUND OF THE INVENTION

FEELD OF THE INVENTION

The present invention relates to a novel pharmaceutical composition for diagnosis or prevention of haemorrhagic fever with renal syndrome caused by viruses in the Hantavinis genus, as well as a nucleocapsid gene of Seoul virus R22 strain, a recombinant expression vector for the said gene, a transformed microorganism with the said recombinant plasmid and a nucleocapsid protein expressed by the said transformant.

DESCRD?TION OF THE PRIOR ART

It has been reported that there are more than ten serologically distinct hantaviruses in the Hantavinis genus of the family Bunyaviridae . Hantaan virus or Seoul virus among such hantaviruses are known as the major aetiological agents of haemorrhagic fever with renal syndrome (HFRS) in Korea. The two viruses contain a tripartite, single-stranded, negative-sense RNA species (S, M and L segments) in common. The S, M and L genome segments encode nucleocapsid protein, glycoproteins Gl and G2, and RNA polymerase, respectively. The nucleocapsid protein and the glycoproteins Gl and G2 are implicated in an immunoreaction.

To develop effective diagnostic agent and vaccines for HFRS, extensive studies have been performed by using viruses per se and proteins implicated in the HFRS immunoreaction. Since the primary condition of HFRS seems to be a cold and the symptoms of HFRS are substantially similar to those suffering from the disease caused by Leptospira or Rickettsia, it is difficult to accurately diagnose the HFRS with clinical observations. The treatment of

HFRS at the primary conditions would prevent such condition from turning fatal. Therefore, it is necessary to make a correct diagnosis of HFRS at the primary conditions.

The correct diagnosis of HFRS can be accomplished by using serological tests. In this assay, blood is collected from a patient twice a week and an increase of titres of anti-hantavirus antibodies is observed by indirect immunofluorescence technique or plaque reduction of neutralization antibody assay. The serological tests are accurate but require at least 7 days. Moreover, these assays are accompanied by cumbersome cell culture.

Many studies have been made to develop alternative diagnostic means of HFRS, while shortening the requirement time for diagnosis and avoiding the cumbersome process of the cell culture. Such studies are focused on the production of viral antigens which react with the anti-hantavirus antibodies in the patient blood. Nucleocapsid protein or Gl or G2 glycoproteins might be considered as the hantavinis antigen for diagnosis of HFRS. The nucleocapsid protein acts as a major antigen at the beginning of the hantavirus infection and induces a large quantity of antibodies in HFRS patients. The antibodies to the nucleocapsid protein are retained for a short duration. In this regard, the nucleocapsid protein are proper as an antigen for diagnosis of HFRS. In contrast, since the antibodies to Gl or G2 glycoproteins can be retained for many years, they are inappropriate as being indicative of the diagnosis of acute HFRS patients. Commercially available antigens used as a diagnostic agent for HFRS are produced through cell culture or enrichment of the hantavirus on mouse brain. However, such cell culture or enrichment requires high costs and have resulted in low yields as well as high danger of viral infections among laboratory workers and animal handlers. Under the circumstances, there have been many studies to massively produce the hantavirus nucleocapsid protein through genetically recombinant technology.

Schmaljohn, etc. of J. Gen. Virol. (1988), 69, 777-786 suggested expression of the nucleocapsid gene of Hantaan virus in insect cells through baculovirus expression system and potential use of the expressed nucleocapsid protein as a diagnostic antigen for HFRS. However, the expressed amount of the nucleocapsid protein is insufficient.

Wang, etc. of 1 Gen. Virol. (1993), 74, 1115-1124 and Gott, etc. of

Virus Res. (1991), 19, 1-16 reported E. coli expression of the nucleocapsid gene of Hantaan virus and potential use of the expressed nucleocapsid protein for diagnosis of HFRS. However, their methods are disadvantageous because of low yield and insufficient purity. Another disadvantage is that the expressed nucleocapsid protein is generated as inclusion body.

It is commonly known that viral vaccines made from attenuated or inactivated whole virus are relatively effective. However, there are several problems in the production of vaccines using attenuated or inactivated virus. First, cultivation of the virus is usually inconvenient, accompaying cell culture.

Second, it is difficult to isolate and purify the virus from cultures in high purity. Third, the possibility to obtain the intact virus is very low because the most of virus is destroyed during purification process. Fourth, the attenuation or inactivation of the purified virus for vaccine are conventionally achieved by formalin treatment or heating. This procedure might lead to denaturation of the vaccine. Fifth, such vaccines are defective in safety which becomes the most considerable factor in production of vaccines.

A first generation vaccine for Hantaan virus has been prepared by using the isolated virus from cerebral fluid of mouse following cultivation of the virus on the mouse brain. However, it is impossible to completely remove the basic myelin protein which is known to cause certain side effect, from the cerebral fluid of mouse. Furthermore, a trace of unidentified proteins derived from mouse brain may exist in the vaccine and as a result, doubts on the safety of the vaccine can arise.

Many researchers have striven to develop cell free production system or in vitro expression system for preparation of the hantavirus antigenic proteins without hazardous proteins or substances. For instance, C. S. Schmaljohn, etc. elucidated nucleotide sequences and gene arrangements of M,

S and L segment genomes of original Hantaan virus 76-118 and observed the preventive capability of Hamster against Hantaan virus following injection of the combined vaccinia gene with M segment encoding glycoproteins Gl and G2 into the animal body. They also reported that S segment encoding nucleocapsid protein increases the preventive capability of glycoproteins.

Further, They managed to express Gl, G2 and nucleocapsid protein of Hantaan virus in insect cells using baculovirus and then addressed the vaccination of animal with the crushed insect cell lysates but failed to observe the formation of neutralization antibodies derived from the nucleocapsid protein of Hantaan virus.

The nucleocapsid protein of hantavirus is a structural protein which is expressed from S segment of RNA of the virus and then exists as a complex with the RNA. Since the nucleocapsid protein constitutes most part of the viral proteins, the purification of the nucleocapsid protein from virus cultures is convenient. However, the studies on the induction of neutralization antibodies of the hantavirus nucleocapsid protein accomplished until now are only the use of vaccinia virus as the expression vector for the nucleocapsid gene and the administration of infected cell lysates including the expressed nucleocapsid protein into animal. The purely isolated nucleocapsid protein of hantavirus has never been applied for a study on the formation of neutralization antibodies and therefore nobody has perceived the hantavirus nucleocapsid protein to be a valuable vaccine for HFRS by generating neutralization antibodies.

Meanwhile, it is very difficult to purify the glycoproteins (Gl and G2) of Hantaan virus and other viruses of the Hantavirus genus and any genetic engineering methods have not made it possible to massively produce the glyco proteins in E. coli, yeast or animal cells.

Despite that many problems may occur when vaccine is prepared from virus per se, all of the currently available vaccines for the prevention of HFRS have been prepared by directly culturing the virus on mouse brain or animal cell and inactivating the purified virus from the cultures with formalin.

The inventors have intensively studied to develop a method which makes it possible to massively produce the antigen for diagnosis of HFRS while ensuring the enhanced safety, diminishing costs and eliminating the tedious and troublesome process of cell culture. The inventors now attained to elucidate the full nucleotide sequence of nucleocapsid gene of Seoul virus R22 and successfully developed a novel expression system capable of massively producing the Seoul virus R22 nucleocapsid protein in a fused form of protein having extra 14 amino acids at amino terminus in E. coli and a method for purifying the nucleocapsid protein. It was now found that the purified nucleocapsid protein produced by the inventors can be used as an active material for the desired rapid, convenient, sensitive, safe HFRS diagnostic preparation. In addition to the use of the present nucleocapsid protein for diagnos is of HFRS patients, it was also found that the present nucleocapsid protein can be also used to determine the formation of antibodies in individuals inoculated with HFRS vaccines. This finding means that the use sphere of the present nucleocapsid protein in the diagnostic preparation for HFRS is wider than that of the current available diagnostic preparations.

The inventors also found that the present nucleocapsid protein of Seoul virus is very useful in the preparation of vaccine for HFRS caused by either

Seoul virus or Hantaan virus. It was reported that the vaccine prepared by inactivating Seoul virus is capable of protecting individuals from even Hantaan virus, in addition to Seoul virus (Yamanishi, et al., Vaccine (1988), 6, 278-282). This report is why the inventors selectively used the nucleocapsid protein of Seoul virus in order to prepare vaccine for prevention of HFRS caused by both Seoul virus and Hantaan virus.

In addition, the inventors found that the vaccine prepared by using the present nucleocapsid protein is considerably superior over the current available vaccines in terms of its efficacy and safety.

SUMMARY OF THE INVENTION

The present invention provides a nucleocapsid gene of Seoul virus R22 having the following nucleotide sequence:

10 20 30 40 50

I I I I I

ATGGCAACTATGGAGGAAATCCAGAGAGAAATCAGTGCTCACGAGGGGCA

60 70 80 90 100 GCTTGTGATAGCACGCCAGAAAGTCAAGGATGCAGAAAAGCAGTATGAAA

110 120 130 140 150 AGGATCCTGATGACTTCAACAAGAGGGCACTGCATGATCGGGAGAGTGTC

160 170 180 190 200

I I I I I GCAGCTTCAATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCCGA

210 220 230 240 250

I I I I I

CAGGATTGCAGCAGGGAAGAATATTGGGCAAGACCGGGATCCTACAGGGG

260 270 280 290 300

I I I I I

TAGAGCCGGGTGATCATCTCAAAGAGAGATCAGCACTAAGCTATGGGAAT

310 320 330 340 350

I I I I I

ACACTGGACCTGAATAGTCTTGACATTGATGAACCTACAGGACAGACAGC

360 370 380 390 400 TGATTGGTTGACCATAATTGTCTATCTGACTTCATTTGTGGTCCTGATCA

410 420 430 440 450

I I I I I TCTTAAAGGCACTGTACATGTTAACAACAAGAGGCAGGCAGACTTCAAAG

460 470 480 490 500

I I I I I

GACAACAAAGGGATGAGGATCAGATTCAAGGATGACAGCTCATATGAGGA 510 520 530 540 550

I I I I I

TGTCAATGGAATCAGAAAACCCAAACATCTGTATGTGTCAATGCCAAACG

560 570 580 590 600

I 1 I I I

CCCAATCCAGCATGAAGGCTGAAGAGATAACACCTGGAAGATTCCGCACT

610 620 630 640 650 i i i i i

GCAGTATGTGGGCTATATCCTGCACAGATAAAGGCAAGGAACATGGTGAG

660 670 680 690 700

I I I I I

CCCTGTCATGAGTGTAGTT∞GTTTTTGGCACTGGCAAAAGATTGGACAT

710 720 730 740 750

I I I I I

CTΛGAATTGAAGAATGGCTTGGTGCACCCTGCAAGTTTATGGCGGAATCT

760 770 780 790 800

I I 1 I I

CCAATTGCTGGGAGTTTATCTGGGAATCCCGTGAATCGTAACTATATCAG

810 820 830 840 850

I I I I I

ACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAGGAATTTCAAGCCC

860 870 880 890 900 i i i i i

TCAGGCAACATGCAAAGGATGCTGGGTGTACACTGGTTGAGCATATTGAG

910 920 930 940 950

I I I I I

TCACCATCATCAATATGGGTGTTTGCTGGGGCCCCTGATAGGTGTCCACC

960 970 980 990 1000

I I I I I CATGTTTGTTTGTCGGAG∞ATGGCTGAGTTAGGTGCCTTCTTTTCTA 1010 1020 1030 1040 1050

I I I I I

TACTTCAGGATATGAGGAACACAATCATGGCTTCAAAAACTGTGGGCACA

1060 1070 1080 1090 1100

I I I I I

GCTGATGAAAAGCTTCGAAAGAAATCATCATTCTATCAATCATACCTCAG

1110 1120 1130 1140 1150 i i i i i

ACGCACACAATCAATGGGAATACAACTGGACCAGAGGATAATTGTTATGT

1160 1170 1180 1190 1200

I I I I I

TTATGGTTGCCTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTGAT

1210 1220 1230 1240 1250

I I I I I

GACATGGACCCAGAGCTTCGTAGCCTGGCTCAGATCCTGATTGACCAGAA

1260 1270 1280 1290

I I I I

AGTAAAAGAAATCTCGAACCAGGAACCTTTGAAACTCTAA

Further, the present invention provides a recombinant expression plasmid including the nucleocapsid gene of Seoul virus R22 of the present invention.

Furthermore, the present invention provides a transformed Escherichia coli with the recombinant expression plasmid of the present invention.

Furthermore, the present invention provides a method for producing a fused form of nucleocapsid protein of Seoul virus R22 in high yield which comprises culturing the transformed E. coli of the present invention on an appropriate medium to express the said nucleocapsid protein, and isolating and purifying the expressed nucleocapsid protein from the cultures.

Furthermore, the present invention provides a highly purified fused form of nucleocapsid protein of Seoul virus R22 having the following amino acid sequence:

10 20 30 40 50

I I I I I

MASMTGGQQMGRGSMATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDP

60 70 80 90 100

I I I I I

DDFNKRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEP

110 120 130 140 150

I I I I I

GDHLKERSALSYGNTLDLNSLDIDEPTGQTADLTI IVYLTSFWLI ILK

160 170 180 190 200

I I I I I

ALYMLTTRGRQTSKDNKGMRIRFKDDSSYEDVNGIRKPKIILYVSMPNAQS

210 220 230 240 250

I I I 1 I

SMKAEEITPGRFRTAVCGLYPAQIKARNMVSPVMSWGFLALAKDWTSRI

260 270 280 290 300

I I I I I

EEWLGAPCKFMAESPIAGSLSGNPVNRNYIRQRQGALAGMEPKEFQALRQ

310 320 330 340 350

I I I I I

HAKDAGCTLVEHIESPSSIWVFAGAPDRCPPTCLFVGGMAELGAFFSILQ

360 370 380 390 400

I I I I I

DMRNTIMASKTVGTADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFMV

410 420 430 440

I I I I

AGKEAVDNFHLGDDMDPELRSLAQILIDQKVKEISNQEPLKL

Furthermore, the present invention provides a - pharmaceutical composition for diagnosis of HFRS comprising the nucleocapsid protein of the present invention in combination with a pharmaceutically acceptable carrier or excipient.

Furthermore, the present invention provides a use of the pharmaceutical composition comprising the nucleocapsid protein of the present invention in determination on the formation of the antibodies to hantaviruses in a subject administered with a vaccine for HFRS.

Furthermore, the present invention provides a diagnostic formulation for HFRS comprising the nucleocapsid protein of the present invention coated on a 96-well plate.

Furthermore, the present invention provides a diagnostic formulation for

HFRS comprising the nucleocapsid protein of the present invention adsorbed on a nitrocellulose membrane.

Furthermore, the present invention provides a vaccine for prevention of HFRS comprising the nucleocapsid protein of the present invention as an active ingredient in combination with a pharmaceutically acceptable adjuvant.

The adjuvant may be selected from a group consisting of aluminium hydroxide gel, chimerosal and tablet gelatin, and a mixture thereof.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows the sites of the chemically snythesized on the nucleocapsid gene of Seoul virus strain R22 nucleocapsid gene.

Fig. 2 is a schematic construction diagram showing a recombinant expression plasmid pET-sNP containing Seoul virus R22 nucleoeapsid gene.

Fig. 3 is a flow diagram showing a purification process for Seoul virus R22 nucleocapsid protein expressed by transformant E. coli BL containing plasmid pET-sNP.

Fig. 4 is (a) polyacrylamide gel electrophoresis and (b) western blots of Seoul virus R22 nucleocapsid protein expressed by transformant E. coli BL containing plasmid pET-sNP.

Fig. 5 is western blots of Seoul virus R22 nucleocapsid protein expressed by transformant E. coli BL containing plasmid pET-sNP using (a) human patient blood and (b) anti-nucleocapsid protein monoclonal antibodies.

Fig. 6 is a photograph showing the reaction of patient serum (P) or healthy subject serum (N) on nitrocellulose membrane adhered by various concentrations of Seoul virus R22 nucleocapsid protein.

Fig. 7 is western blots of nucleocapsid proteins expressed by several transformant E. coli strains containing different vectors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention was accomplished through the amplification of the fully elucidated nucleotide sequence of a nucleocapsid gene of Seoul virus

R22, the preparation of a recombinant expression plasmid, the production of a transformed E. coli, the cultivation of the transformant, the isolation and purification of the expressed nucleocapsid protein, and the evaluation of the diagnostic agent and vaccine comprising the nucleocapsid protein on efficacy and safety. A large amount of the nucleocapsid gene of Seoul virus R22 to be cloned into an expression vector was prepared through twice polymerase chain reactions using three primers having the following nucleotide sequences, respectively:

NP 1 (17MER): 5'-TAGTAGTAGACTCCCTA-3'

NP 2 (23MER): 5'-CCAGATCTATGGCAACTATGGAG-3'

NP 3 (22MER): S'-GGAATTCTTAGAGTTTCAAAGG-S'

EcoR I

The first polymerase chain reaction used the designated NPl and NP3 primers to amplify the nucleocapsid gene of Seoul virus R22. The second polymerase chain reaction used the designated NP2 and NP3 primers to introduce recognization sites for Bg l and 20 EcoRI into the nucleocapsid gene, which will be thereby cloned into a commercially available expression vector pET-3a (Stratagene, cat#211621).

Since the vector pET-3a used for the cloning of the nucleocapsid gene of the present invention includes slO sequence, the nucleocapsid protein is expressed as a form of a fused protein having additional 14 amino acids at the amino terminus.

Fig 2. illustrates a whole process for constructing a recombinant plasmid for expression of the nucleocapsid gene of the present invention by cloning the said gene into the vector pET-3a. The resulting recombinant plasmid was designated as pET-sNP.

A novel transformed microorganism was produced by introducing the plasmid pET-sNP of the present invention into a host cell, E. coli BL21(DE3) (Stratagene, cat#211621) through electric shock and was proved as being capable of massively producing the desired nucleocapsid protein. This microorganism, referred to as E. coli BL(pET-sNP), was deposited with the Korean Culture Center of Microorganisms under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on September 10, 1997 and the accession number was designated as KFCC-10111.

The nucleotide sequencing of the nucleocapsid gene of Seoul virus R22 was carried out by using Auto Reader Sequencing Kit (Pharmacia Co., cat#27-2690-20, cat#l 8-1033-13) in accordance with a known dideoxy method

(PNAS 74, p5463, 1977). Then, the recombinant expression plasmid pET-sNP was used as a template and the synthetic fragments having the following nucleotide sequences were used as a primer:

PI: 5' GGCTAGCATGACTGGTGG-3'

P2: CTATCTGACTTCATTTGTGG

P3: ACCCAAACATCTGTATGTG

P4: GTTTATCTGGGAATCC

P5: GAGGAACACAATCATGG

P6: CCATTATTATCATAGC

P7: TGATTGTGTGCGTCTGAGGT

P8: ATTCTTCAATTCTAGATGTC

P9: CAGTGCCTTTAAGATGAT

In order to ensure the accuracy of the nucleotide sequencing, the nucleocapsid gene was twice analysed from 5'-end to 3'-end and, reversely, from 3'-end to 5'-end. The elucidated nucleotide sequence of the nucleocapsid gene of Seoul virus R22 in comparison with the known nucleotide sequence of the nucleocapsid gene of Seoul virus SRl l(Virology 176, ppl 14-125, 1990) is as follows:

10 20 30 40 50

A∞CAACT^ABGAAATCCA GAGAΛTCAG∞TdACGAGGGGCA' R-22 .SEQ

ATGGCAACTATGGAΠGAAATCCAGAGAGAAATCAGTGCTCACGAGGGGCA SR- 11 . SEQ

60 70 80 90 100 GCTTGTGATAGCACGCCAGAAIUGTCAAGGATGCAGAAAAGCAGTATGAΔA R-22 .SEQ

GCTTGTGATAGCACGCCAGAAIBGTCAAGGATGCAGAAAAGCAGTATGAGA SR- 11 .SEQ

110 120 130 140 150

AGGATCCTGAVGACΓΓSMCAAGAGGGCACTGCAMTC∞AGAGTGTC R-₂₂ .SHJ

AGGATCCTGATGACTTI.IAACAAGAGGGCACTGCATGATCGGGAGAGTGTC SR-11.SEQ

160 170 180 190 200

I I I I I

GCAGCTTCAATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCCGA R-22 .5SEQ GCAGCTTCAATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCCGA SR- 11 .SEQ

210 220 230 240 250

CAGGATTGCAGCAGGGAAGAAΪΪATΪΪGGGCAklGACCGGGATCCTACAGGGG R-22 .SEQ

CAGGATTGCAGCAGGGAAGAASATS!GGGCAI8GACCGGGATCCTACAGGGG SR-11 . SEQ

260 270 280 290 300

TAGAGCCffcGTGATCATCτ5!AAl-feA(aAGATCAGCACTAAGCTAΪ!GGGAAT R-22 .SEQ

TAGAGCClUGGTGATCATCTnAAfflGAElAGATCAGCACTAAGCTAfflGGGAAT SR-11 .SEQ

310 320 330 340 350

I I I I I

ACACTGGACCTGAATAGTCTTGACATTGATGAACCTACAGGACAGACAGC R-22.SEQ

ACACTGGACCTGAATAGTCTTGACATTGATGAACCTACAGGACAGACAGC SR-11.SEQ

360 370 380 390 400

TGATT« rGACCATAATTGTCTATCTGAθ ^,CATTΪ-GTGGTCCΪΪGATCA R-22 .SEQ

TGATTGGBh-GACCATMTTGTCTATCTGAciu-CATTSGTGGTCCfflGATCA SR- 11 . SEQ 410 420 430 440 450

_TCτAAGGc cTGTACATG™CAACAl GGlSAGGC GACITCAAAG^, R-22 .SHI

TCTT^GGCACTGTACATGTTAACAACASGAGGΪSAGGCAGACTTCAAAG SR- 11 . SEQ

460 470 480 490 500 _β ι i i i i

GACAACAAEIGGGATGAGGATCAGATTCAAGGATGACAGCTCATATGAGGA R-22 .SEQ

GACAACAAIUGGGATGAGGATCAGATTCAAGGATGACAGCTCATATGAGGA SR- 11 .SEQ

510 520 530 540 550

TGTCMTGGATCAGAAAΠCCCAAACATCTGTATGIT.TCAVTGCCAAACG R-22 .SEQ TGTCAATGGAATCAGAAAIRCCCAAACATCTGTATGTGTCAATGCCAAACG SR-11 . SEQ

560 570 580 590 600

CCCAATCCAGCATGAAGGCTGAAGAGATAACACCΪSGGAAGATTCCGCACT R-22 .SEQ

CCCAATCCAGCATGAAGGCTGAAGAGATAACACAGAAGATTCCGCACT SR-11 .SEQ

610 620 630 640 650

GCAGTATGTGGISCTATATCCTGCACAGATAAAGGCAAGGAARATGGTSAG R-22 .SEQ

GCAGTATGTGATATATCCTGCACAGATAAAGGCAAGGAA-IATGGTBAG SR- 11. SEQ

660 670 680 690 700

CCCTGTCATGAGTGTAGTTGGGTT!1TTGGCACTGGCAAAAGAΠTGGACAT R-22 .SEQ

CCCTGTCATGAGTGTAGTTGGGTTSTTGGCACTGGCAAAAGASTGGACAT SR-11 . SEQ

710 720 730 740 750

Cl-AGMTTG G TGC^T-fcGTGCACCCTG^AAl&rT^TGGCGGAElTCT R-22 .SEQ ^GMTTGMG TGGCTtiGTGCACCCTGCAAEh fflATGGCGGAfflTCT SR- 11 . SEQ

760 770 780 790 800

CCMTTGCIGGGAGTTTATCTGGG TCC GTGMTCGTI-IACTATATCAG R-22 .SEQ

CrLATTGCCGGGAGTTTATCTGGGAATCCΪ.GTGAATCGTfflACTATATCAG SR-11 .SEQ 810 820 830 840 850

I I I I I

ACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAGGAATTTCAAGCCC R-22.SEQ

ACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAGGAATTTCAAGCCC SR-11.SEQ

860 870 880 890 900

TCAGGC CATJfc AGGATGCTGGfϊ^GTACACTlfcTTGASfcATATTGAG R-22 .SEQ

TCAGGCMCATBCMA∞ATGCTGG^GTACACT^TTGAIIICATATTGAG SR-11 . SEQ

910 920 930 940 950

I I I I I

TCACCATCATCAATATGGGTGTTTGCTGGGGCCCCTGATAGGTGTCCACC R-22 .SEQ TCACCATCATCAATATGGGTGTTTGCTGGGGCCCCTGATAGGTGTCCACC SR- 11 . SEQ

960 970 980 99 1000

MCATC_1-ΪTTGTTTGTCGGAGGGATGGCTGA AGGTGCCTTCTTTTCTA R-22 .SEQ CATC TTGTTTGTCGGAGGGATGGCTGAl- AGGTGCCTTCTTTTCTA SR-11 .SEQ

1010 1020 1030 1040 1050

I I I I I

TACTTCAGGATATGAGGAACACAATCATGGCTTCAAAAACTGTGGGCACA R-22.SEQ TACTTCAGGATATGAGGAACACAATCATGGCTTCAAAAACTGTGGGCACA SR-11.SEQ

1060 1070 1080 1090 1100

I I 1 I I

GCTGATGAAAAGCTTCGAAAGAAATCATCATTCTATCAATCATACCTCAG R-22.SEQ

GCTGATGAAAAGCTTCGAAAGAAATCATCATTCTATCAATCATACCTCAG SR-11. EQ

1110 1120 1130 1140 1150

I I I I I

ACGCACACAATCAATGGGAATACAACTGGACCAGAGGATAATTGTTATGT R-22.SEQ ACGCACACAATCAATGGGAATACAACTGGACCAGAGGATAATTGTTATGT SR-11.SEQ

1160 1170 1180 1190 1200

I I I I I

TTATGGTTGCCTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTGAT R-22.SEQ TTATGGTTGCCTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTGAT SR-11.SEQ 1210 1220 1230 1240 1250

GACATGGASicCAGAGCTTCGTAGCCTGGCTCAGATCtrGATTGACCAGAA R-22 .SEQ

GACATGGAΠCCAGAGCTTCGTAGCCTGGCTCAGATCΪSTGATTGACCAGAA SR- 11. SEQ

1260 1270 1280 1290 AGτBME!GAMTCTCffl CCAGGMCCTπTGAMfflτB!TAA R-22 .SEQ

ΛGTffl ^AMTCTC^CCAGGMCCTl^GAMilτEITAA SR-11 .SEQ

in which the reversed A, T, G, and C indicate the different nucleotides between nucleotide sequences of nucleocapsid genes of Seoul virus R22 and Seoul virus SR11. A comparison of the two nucleotide sequences reveals that 1241 nucleotides are same and thus the identity is calculated as 96.2%. In addition, upon comparison of the two presumed amino acid sequences from the Seoul virus R22 and Rl l nucleotide sequences as illustrated below, it reveals that 423 amino acids are same and thus the identity is calculated as 98.6%:

10 20 30 40 50

MATMEE IQRE I SAHEGQLV I QKVKDAEKQYEIΦPI)I iB KRALHDRESV R-22 .PRO

MATMEE IQREISAHEGQLVIARQKVKDAEKQYEKDPDDINKRALHDRESV SR-11 .PRO

60 70 80 90 100

I I I I I

AΛSIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGN R-22.PRO AASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEPGDHLKERSALSYGN SR-11.PRO

110 120 130 140 150 i i i _ i i

TLDLNSLI)IDEPTGQTADWLTI IVYLTSFWilILKALYMLTTRGRQTSK R-22.PRO

TLDLNSLDIDEPTGQTADWLTIIVYLTSFWlBl ILKALYMLTTRGRQTSK SR-11.PRO 160 170 180 190 200

I I I I I

DNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT R-22.PRO DNKGMRIRFKDDSSYEDVNGIRKPKHLYVSMPNAQSSMKAEEITPGRFRT SR-11.PRO

210 220 230 240 250

I I I I I

10 AVCGLYPAQIKARNMVSPVMSWGFLALAKDWTSRIEEWLGAPCKFMAES R-22 .PRO

AVCGLYPΛQ IKARNMVSPVMSWGFLALAKDWTSRIEEWLGAPCKFMAES SR-11 .PRO

260 270 280 290 300

BlAGSLSGNPVNRfflYIRQRQGALAGMEPKEFQALRQHlUKDAGCTLVEHIE R-22 .PRO

IIAGSLSGNPVNRIUY IRQRQGALAGMEPKEFQALRQHBKDAGCTLVEHIE SR-11 .PRO

20

310 320 330 340 350

I I I I I

SPSSIWVFAGAPDRCPFΓCLFVGGMAELGAFFSILQDMRNTIMASKTVGT R-22.PRO SPSSIWVFAGAPDRCPPTCLFVGGMAELGAFFSILQDMRNTIMASKTVGT SR-11.PRO

360 370 380 390 400 30 i i i i i

ADEKLRKKSSFYQSYLRRTQSMGIQLDQRI IVMFMVAWGKEAVDNFHLGD R-22.PRO

ADEKLRKKSSFYQSYLRRTQSMGIQLDQRIIVMFMVAWGKEAVDNFHLGD SR-11.PRO

410 420 430

DMDPELRSLAQ IL IDQKVKE I SNQEPlKL R-22 .PRO

40

DMDPELRSLAQ IL IDQKVKELSNQEPiϊ L SR-11 .PRO

in which the reversed characters indicate the different amino acids between amino acid sequences of nucleocapsid proteins from Seoul virus R22 and SR11. The fragments which serve as a primer for sequencing the nucleocapsid gene of Seoul virus R22 were chemically synthesized on the basis of the known nucleotide sequence of the nucleocapsid gene of Seoul virus SR-11. Fig. 1 shows the location of the chemically synthesized primers on the nucleocapsid gene of Seoul virus R22.

The culture of the transformed E. coli of the present invention can be performed according to methods conventionally known in the art of a genetic engineering technology. The high purification of the expressed nucleocapsid protein was accomplished through ammonium sulfate precipitation, gel filtration and phenyl sepharose column chromatography. As illustrated in Fig. 3, subsequent polyacrylamide gel electrophoresis and western blotting were carried out to affirm the purified nucleocapsid protein. The results of polyacrylamide gel electrophoresis and western blotting shown in Figs. 4 and 5, respectively, prove that the purified substance corresponds to the nucleocapsid protein. In addition, the purity of the nucleocapsid protein was evaluated as at least 90% from the result of polyacrylamide gel electrophoresis.

The purified nucleocapsid protein of the present invention includes additional 14 amino acids(Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-

Gly-Ser) at the N-terminus as compared to the corresponding naturally occurred nucleocapsid protein. The additional amino acid sequence is derived from slO leader sequence located at the front of BamHl site, cloning site of plasmid pET-3a. The molecular weight of the present nucleocapsid protein was measured as approximately 50 kDa.

An alternative recombinant plasmid, designated as pKK-sNP, for expression of the nucleocapsid protein gene of Seoul virus R22 was constructed by the inventors. The construction of the plasmid pKK-sNP is for the purpose of inspecting the influence of a cloning vector on the expression of the nucleocapsid protein gene of Seoul virus R22. The procedure for constructing the plasmid pKK-sNP was similar to that of the plasmid pET-sNP except that pKK223-3 having the strong tac promoter (Pharmacia Biotech, cat#27-4935-01) was used as a cloning vector instead of pET-3a. It was found that the pET-3a express the nucleocapsid protein of Seoul virus R22 much higher than the pKK223-3. It is comprehended that such result is because the fused form of the nucleocapsid protein with additional 14 amino acids at the N-terminus allows the expressed nucleocapsid protein to be more stable in E. coli or because slO leader sequence existing in pET-3 plays a role in increasing either the transcription efficiency of the cloned nucleocapsid protein gene or the translation efficiency in the protein synthesis from the transcript mRNA.

For the evaluation of the nucleocapsid protein expression capability of the plasmid pET-sNP of the present invention, another plasmid, designated as pET-NP was constructed by cloning the nucleocapsid gene of prototype

Hantaan virus 76-118 (Virology 155, pp633-643, 1986) into pET-3a in accordance with the same procedure as used in the construction of pET-sNP of the present invention. The two expressed nucleocapsid proteins of Seoul virus R22 and Hantaan virus 76-118 were separately detected by western blotting using monoclonal antibodies to the Hantaan virus nucleocapsid protein.

The results are shown in Fig. 7. It can be seen that the expressed Seoul virus R22 nucleocapsid protein is twice as high as the expressed Hantaan virus 76-118 nucleocapsid protein. The results are likely attributed to the higher stability of the Seoul virus R22 nucleocapsid protein than that of the

Hantaan virus 76-118 nucleocapsid protein.

An assay on a diagnostic efficacy of the Seoul virus R22 nucleocapsid protein of the present invention was performed according to conventional methods. The nucleocapsid protein coated on 96-well plate or nitrocellulose membrane was reacted with patient serum or vaccinee serum. In order to ensure the accuracy of the assay, the blood collected from the patient was re-assayed by an indirect immunofluorescence antibody assay. The results are shown in Fig. 6. It can be seen that the patient serum was accurately detected by both of the Seoul virus R22 nucleocapsid proteins coated on 96-well plate or nitrocellulose membrane. Also it was appreciated that the Seoul virus R22 nucleocapsid protein can be used to examine the formation of the antibodies in vaccinated subjects with HFRS vaccines.

The purified nucleocapsid protein of Seoul virus R22 of the present invention is mixed with conventional adjuvants, especially aluminium hydroxide gel, prior to its use as a formulated vaccine for HFRS. The preferred amount of the aluminium hydroxide gel used is less than 0.625 mg/ 0.5 ml of vaccine solution. In addition to the adjuvant, chimerosal and purified gelatin may be used as a preservative in amount of 0.01% (w/v) and a stabilizing agent in amount of 0.02% (w/v), respectively. Also, 0.01% (w/v) of Tween 80 can be added.

The invention will now be explained in details with reference to the following illustrative Examples.

Example 1

Isolation of genomic RNA from Seoul virus R22 and synthesis of cDNA

100 μl of culture solution of Seoul R22 virus was mixed with 400 μl of Solution A (4.2 M guanidine isothiocyanate, 25 ml Tris-HCl, pH 8.0, 0.5% Sarkosyl, 0.7% β-mercapto ethanol) and then with 50 μl of Solution B (1 M Tris-HCl, pH 8.0, 0.1 M EDTA, 10% SDS). After incubation of the resuting solution at 65 °C for 5 minutes, it was mixed with an equal volume of a mixed solution of phenol and chloroform (1 : 1). After another incubtion at 6 5°C for further 30 minutes, the mixture was centrifuged. The supernatant was taken, and the remaining solution was mixed with further 300 μl of Solution A, then incubated at 65 ^°C for 5 minutes. Following centrifugation of the solution, the supernatant was taken and combined with the first supernatant. The total supernatant was extracted with a mixed solution of phenol and chloroform (1:1) and then with chloroform. The extracted solution was mixed with 1/10 volume of 3 M sodium acetate and two volumes of isopropyl alcohol and the resulting mixture was kept at -20 ^°C for 16 hours. The solution was then centrifuged at 12,000 rpm for 15 minutes, and the precipitate was washed with 70% ethyl alcohol twice and dried. The dried precipitate was dissolved in 10 μl of sterilized distilled deionized water which does not contain ribonuclease at all and the resulting solution was kept at 4°C . 5 μl of the purified genomic RNA was used to synthesize cDNA. The synthetic reaction was carried out at 37 °C for 1 hour under the conditions indicated in Table 1 below.

Table 1

Example 2

Amplification of nucleocapsid gene by the polymerase chain reaction

The nucleocapsid gene to be cloned into a known plasmid pET-3a was amplified by the polymerase chain reaction (PCR) with the primers illustrated above. The DNA Thermal Cycler 480 (Perkin Elmer) was used for the PCR. The reaction conditions and procedures are described in Table 2 below.

Table 2

5 μl of cDNA solution prepared by Example 1 was first amplified by the polymerase chain reaction with NP1 and NP3 primers. 5 μl of the first reacted cDNA solution was continuously subject to the second polymerase chain reaction using NP2 and NP3 primers which contain recognition sites of

BgAl (AGATCT) and EcoRI (GAATTC) at 5' ends, respectively.

Example 3

Construction of expression plasmid pET-sNP

The process for the cloning of the nucleocapsid gene was depicted in Fig. 2. The two double-stranded DNA fragments amplified in Example 2 were characterized by agarose gel electrophoresis and then the DNA fragment containing 1.3 kb of nucleocapsid protein was recovered using GENECLEAN kit. The recovered DNA fragments were digested with restriction enzymes BgUl and EcoRI, extracted once with a mixed solution of phenol and chloroform (1:1) and dissolved in 20 μl of sterile distilled water. Likewise, 5 μg of plasmid pΕT-3a as a cloning vector was digested with BgM and EcoRI, heated to 70 "C for 10 minutes in presence of 5 mM of ΕDTA, extracted once with a mixed solution of phenol and chloroform (1 : 1) and dissolved in 20 μl of sterile distilled water. 5 μl of the vector was mixed with - 10 μl of the restriction enzyme-cleaved nucleocapsid DNA and the ligation reaction was carried out at 25 °C for 3 hours in presence of T4 DNA ligase to form the desired expression plasmid pET-sNP.

Example 4

Construction of plasmid pKK-sNP

As primers, random 6-mers were used to synthesize cDNA from genomic RNA of Seoul virus R22. The first amplification of DNA was carried out by using the cDNA as template with NP1 and NP3 primers illustrated above. The second amplification of the DNA fragment was carried out by using the first amplified fragment as template with the following NP4 and NP5 primers which contain recognization sites of restriction enzymes EcoRI (GAATTC) and Sail (GTCGAC) for cloning into pKK223-3, respectively:

NP 4 (23MΕR): 5 -CCGAATTCATGGCAACTATGGAG-3'

EcoR I

NP 5 (23MER): 5 -GCGTCGACTTAGAGTTTCAAAGG-3'

Sai l

The amplified nucleocapsid DNA fragment recovered from agarose gel was treated with Klenow enzyme to make both ends blunt and then the resulting blunt-ended DNA was treated with EcoRI to make one end cohesive. The vector pKK223-3 was digested with EcoRI and Smaϊ and ligated with the EcoRI-cleaved nucleocapsid gene. The resulting recombinant plasmid pKK-sNP was introduced into E. coli JM105. It was found that pKK223-3 contains two Sail sites. This is why Smal site was used instead of Sail for the construction of pKK-sNP.

Example 5

Production of transformed E. coli

An electric shock was used for the transformation and E. coli BL21(DE3) was used as a host cell for pET-sNP. 20 ml of LB medium was inoculated with a plate culture of the microorganism E. coli BL21(DE3) and a shake culture was carried out at 37 TJ for 18 hours. 1 L of freshly made LB medium was inoculated with the first shake culture and a second shake culture was carried out at 37^°C until the optical density of 0.5-0.8 at 600 nm was reached. The resulting culture solution stood at 0^°C for 20 minutes and centrifuged to recover the cells. The recovered cell pellet was successively washed once with 1 L of cold sterile distilled water, once with 0.5 L of cold sterile distilled water and once with 20% glycerol. The cells were resuspended in 3 ml of 10% glycerol, divided into aliquots and kept at -70 TJ . Gene-Pulser (Bio-Rad) was used in electrical shock. A mixture of 40 μl aliquots of cells with 2 μl of DNAs was placed into cuvette with 0.2 cm of electrode gap. After one electric shock was pulsated on the cuvette following the adjustment of the Gene-Pulser at 25 μF of capacitance, 200 Ω of electric resistance and 12.5 kV/cm of electric field strength, immediately 1 ml of SOC medium (2% bactotrypton, 0.5% yeast extract, 10 ml NaCl, 2.5 mM KC1, 10 mM MgCl₂, 10 mM MgS0₄, 20 mM glucose) was added. After a shake culture at 37 J for 1 hour, 0.1 ml and 0.9ml of cell cultures were sprayed on two LB medium agar plate with 50 μg/ml of ampicillin, respectively, and cultured in an incubator at 37TJ for 12 to 18 hours. A loop of cells picked from the agar plate medium was put into 80 μl of cracking buffer (0.05 M Tris, pH 6.8, 1% SDS, 2 mM EDTA, 0.4 M sucrose, 0.01% bromophenol blue) and was made disperse by a vortex mixer. The disperse solution was centrifuged at 12,000 m for 15 minutes. The supernatant was subjected to an agarose gel electrophoresis to screen pET-sNP. The pET-sNP was reconfirmed by a digestion with restriction enzymes.

Example 6

Culture of transformant

The transformed cells were cultured overnight on LB medium (0.5% yeast extract, 1% bactotrypton, 1% NaCl, pH 7.0) with supplementary 0.5% glucose and 100 μg/ml of ampicilin. 1 L of the freshly made LB medium was inoculated with 5% overnight cell culture and was shaken at 37°C with the velocity of 200 φm. Once the optical density of 0.5 to 0.8 at A₆₀o was reached, 0.1 to 2 mM D?TG was added to the culture solution and cultured for further 4 to 8 hours. The cells were recovered from the culture solution by centrifugation and washed once with 0.8% NaCl.

Example 7

Isolation and purification of nucleocapsid protein

A method for purifying the expressed nucleocapsid protein from a transformed E. coli with pET-sNP was illustrated in Fig. 3. The recovered cells were suspended in 50 to 100 ml of TE buffer solution (50 mM Tris, 1 mM EDTA, pH 8.0) and crushed by a sonicator. The sonication was continued until the concentration of the proteins in the crushed solution was no longer increased by a Bradford assay. The crushed solution was centrifuged at 8,000 x g for 1 hour. The amount of ammonium sulfate to be 25% to 50% of the saturated concentration was dissolved in the recovered supernatant. The resulting solution stood at room temperature for 1 hour and centrifuged at 8,000 x g for 30 minutes. The supernatant was discarded and the precipitate was resuspended in 20 ml of TE buffer solution. The suspension was dialyzed twice in 2 L of TE buffer solution each for two hours to remove ammonium sulfate. The dialyzed solution was applied to gel filtration with Sepharcryl S200 (Pharmacia, Co), and then hydrophobic interaction chromatography with phenyl sepharose CL-4B resin (Pharmacia, Co). For loading of the samples, a solution of 40 mM KH₂P0₄/Na₂HP0₄ (pH 8.0) containing 0.6 M of ammonium sulfate was used, while, upon elution, a solution of 40 mM KH₂P04 Na₂HP0₄ (pH 8.0) without ammonium sulfate was used. Thereby, most nucleocapsid proteins were flown through, with most other proteins originated from E. coli being eliminated. The eluted nucleocapsid proteins were further dialyzed in PBS buffer solution and concentrated by Centricon or Centriprep (Amicon, Co.). All of the step-wise purified proteins were analyzed by polyacrylamide gel electrophoresis and western blotting (Fig. 4). The purity of final protein assumed to be higher than 90%.

Example 8

Western blotting of nucleocapsid protein

The purified proteins expressed from the transformed cells of the present invention were subjected to a polyacrylamide gel electrophoresis. The proteins on polyacrylamide gel were then transferred to nitrocellulose membranes and western blotting was carried out by using HFRS patient sera and anti-nucleocapsid protein monoclonal antibody ht9040. The membranes were reacted with 5% PBS solution (8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HP0₄, 0.24 g KH₂P0₄, pH 7.4, per 1 L of PBS solution) containing skim milk for 30 minutes to permit only proteins having specificities to be adhered and then reatcted with the above monoclonal antibody ht9040 at room temperature for more than one hour. The membranes were washed three or four times with PBST solution (PBS + 0.5% Tween 20) each for 5 to 10 minutes to remove the unadhered antibodies. The washed membranes were immersed into PBS solution containing 5% skim milk and appropriately diluted peroxidase-labelled anti-mouse immunoglobulin G and shaken for 1 hour. The membranes were washed further three or four times with PBST solution each for 5 to 10 minutes and stained with 4-chloro-l-naphtol. The desired nucleocapsid protein specific band (about 50 Kd) was exhibited on the expected spots (Fig. 5).

Example 9

Diagnostic assay on the purified nucleocapsid protein coated on 96-well plate by ELISA

An enzyme-linked immunoadsorbent assay (ELISA) was carried out to determine whether the Seoul virus nucleocapsid protein expressed by the transformed E. coli of the present invention is capable of recognizing HFRS patient sera. Twenty (20) HFRS patient sera and four (4) normal sera as control were tested. Each microtitre well in EIA/RIA 96-well plate was coated with 100 μl of solution of the nucleocapsid protein (100 ng/well) in coating buffer (50 mM NaHC03, pH 9.0) at normal temperature for 1-2 hours and washed twice with PBS buffer. 100 μl of patient or normal sera diluted with PBS was placed into each microtitre well and reacted for 1 hour. The plate was washed twice with PBST buffer and reacted with 1/1000 diluted peroxidase-labelled anti-mouse immunoglobulin G for 1 hour. Subsequently, the plate was washed twice with PBST buffer and 100 μl of 0.1 M citrate-phosphate buffer (pH 4.9) containing 1 mg/ml of OPD (o-phenylendiamine dihydrochloride) and 0.03% H₂0₂ was placed into each microtitre well. After the plate was kept at room temperature for 20 to 30 minutes in the dark, the reaction was stopped by placing 50 μl of 1 M sulfuric acid into each microtitre well and the absorbance at 490 nm was measured by ELISA Reader. The ELISA titres were defined as the reciprocal of the maximum dilutions of sera that generated absorbance readings higher than 0.2. The results were shown in Table 3 below. It should noted from Table 3 that there are remarkable differences between ELISA titres of IgGs or IgMs from HFRS patients and those from normal humans with the expressed nucleocapsid protein. This indicates that ELISA using the expressed nucleocapsid protein of the present invention is very useful in the accurate diagnosis of HFRS. In order to enhance the accuracy of ELISA, all sera were subjected to indirect immunofluorescence assay. From Table 3, it should be noted that the results obtained by ELISA are completely consistent with those obtained by indirect immunofluorescence antibody assay.

Table 3

ELISA reactivities of HFRS patient sera and negative control human sera with the expressed nucleocapsid protein

* "+" and "-" signs represent test results positive and negative, respectively.

Example 10

Diagnostic assay by the use of the purified nucleocapsid protein-adhered nitrocellulose membrane

ELISA employing 96-well plate is useful in diagnosing many patients but requires the instrument ELISA reader. There is a need to conveniently diagnose a few HFRS patients occurred at rural or small areas.

The purified nucleocapsid proteins were diluted in PBS to make the concentrations of 100, 50, 25, 13 and 6 μg/ml. The nitrocellulose membranes were immersed into the diluted solutions and the reactions were allowed by gently shaking it for 1 hour. The membranes were blocked by using PBS buffer solution with skim milk dissolved as 5%, dried at 30 TJ for 4 hours and reacted with 1/500 dilution of patient sera in PBS buffer at normal temperature for 1 hour. Then, the membranes were washed twice with PBST solution (PBS, 0.5% Tween 20) and were reacted with 1/1000 dilution of second peroxidase-labelled anti -human antibodies in PBS for 1 hour. Subsequently, the membranes were washed twice with PBST solution and were stained with coloring agent [25 ml of 0.1 M Tris, pH 7.4, 25 mg of

4-chloro-l-naphtol (5 mg/ml in methanol), 3 μl of H₂0₂, 20 ml of distilled water]. When the color was appeared on the membranes, the reaction was allowed to be stopped and the membranes were washed with distilled water. It was observed that 13 μg/ml to 100 μg/ml of nucleocapsid protein solution resulted in the conspicuous appearance of the color by response with patient sera, while even 100 μg/ml of nucleocapsid protein solution did not result in the appearance of the color by respond with any normal human sera.

Table 4 below shows the serological reactivities of 100 μg/ml or 10 μ g/ml of nucleocapsid protein solution adhered on nitrocellulose membranes with twenty patient sera and four normal human sera. All sera were affirmed by performing indirect immunofluorescence antibody assay.

Table 4

* Number of "+" signs represents the thickness of colors.

** "+" and "-" signs represent test results positive and negative, respectively.

Example 11

Assay on the reactivity of the purified nucleocapsid protein with HFRS vaccinee serum

Ten individuals were vaccinated with current commercially available vaccine, Hantavax(Korean Green Cross, Co.) according to the vaccination schedule recommended by the producer, and were bled one month from the last vaccination. The reactivities of the purified nucleocapsid protein of Seoul virus R22 with pre-inoculation sera or post-inoculation sera were observed by performing similar procedures to Examples 8 and 9 except for using vaccinee sera instead of patient sera. The concentration of the nucleocapsid protein adhered on nitrocellulose membrane was 10 μg/ml. The results are shown in Table 5 below. It is noted from Table 5 that the results obtained by the use of nitrocellulose membrane are completely consistent with ELISA results. The results in Table 5 indicate that the purified nucleocapsid protein of the present invention is very useful in determining whether HFRS vaccine induced antibodies following its vaccinations.

Table 5

* Number of "+" signs represents the thickness of colors, and

"+" and "-" signs represent test results positive and negative, respectively.

Example 12

Assay on the efficacy of the vaccine prepared from the purified nucleocapsid protein 0.5 ml of the purified nucleocapsid protein in Example 7 was mixed with 0.625 mg of aluminum hydroxide gel as adjuvant and the mixture stood at 4TJ for 15 days. Thereafter, 0.01% (w/v) chimerosal and 0.02% gelatin were added to the mixture to prepare the final test vaccine.

Ginea pigs were used for the assay of the induction of neutralization antibodies of the vaccine. The current commercially available vaccine prepared by inactivating the virus cultured on rat brain was used as control. The concentrations of the antigen to be inoculated on the experimental animals were 10 μg/0.5 ml, 20 μg/0.5 ml and 40 μg/0.5 ml. The sera of guinea pigs inoculated with test vaccine and control vaccine were subjected to plaque reduction neutralization test to assay the immune efficacy of the vaccines. The plaque reduction neutralization test was performed as follows:

1) In order to obtain antibodies for the plaque reduction neutralization test, guinea pigs were subcutaneously inoculated three times at an interval of 10 days with three different concentrations of test vaccines and control vaccines (0.5 ml/inoculation).

2) Sera from guinea pigs were inactivated at 56TJ for 30 minutes and were diluted 1:10, 1 :20, 1 :40 and 1:80 in medium (MEM + M199 = 1 :1) containing 3% fetal bovine serum.

3) The diluted sera were mixed with equal volumes of Hantaan virus 76-118 diluted to be 70 PFU/culture vessel (diameter of 6 cm) in test tubes and reacted at 37TJ for 1 hour.

4) The preparative monolayer of Vero E6 cell in 6 cm culture vessel was inoculated with 0.2 ml of the above mixed solution and the reaction was allowed at 37TJ for 90 minutes. 5) Inoculated medium was removed and 5 ml of the first agarose overlay was formed on each culture vessel. The composition of the first agarose overlay is as follows:

Ml 99 medium 50 ml

Fetal bovine serum 10 ml

7% NaHC0₃ 3 ml

Agarose 0.8 g Distilled water 50 ml

The Ml 99 medium contains no phenol red and the fetal bovine serum was heated to 56 TJ for 30 minutes.

6) After the first agarose overlay was completed, inoculated cells were cultured at 37 TJ for 10 to 11 days.

7) 3 to 3.5 ml of the second agarose overlay was formed on each culture vessel. The composition of the second agarose overlay is as follows:

M199 50 ml

400 mM MES^* 10 ml

5% BSA 5 ml

0.6% Neutral Red 1 ml IN NaOH 1.5 ml

Agarose 0.7 g

Distilled Water 32.5 ml

*(2-(N-Moφholino)ethane sulfonic acid, monohydrate) 8) After the second agarose overlay was completed, it was cultured for 3 days and the number of plaques with diameter of about 2 mm was counted.

Table 6

It should noted from Table 6 above that the induction of neutralization antibodies of the test vaccine prepared from the purified nucleocapsid protein at concentration of 40 μg/0.5 ml and 20 μg/0.5 ml are much higher than the control vaccine, while the inductions of neutralization antibodies of the test and control vaccines at concentration of 10 μg/0.5 ml are equal. The results indicate that the vaccine prepared from the nucleocapsid protein of Seoul virus R22 according to the present invention exhibits excellent immunogenicity against HFRS as compared with the current available vaccines. Especially, since the vaccine of the present invention is prepared from the single nucleocapsid protein, rather than the virus per se which is believed to include toxical materials, it is apparent that the vaccine of the present invention is very superior to the current available vaccines in terms of safety.

Claims

WHAT IS CLAIMED IS:

1. A Seoul virus R22 nucleocapsid gene having the following nucleotide sequence: 10 20 30 40 50

I I I I I

ATGGCAACTATGGAGGAAATCCAGAGAGAAATCAGTGCTCACGAGGGGCA

60 70 80 90 100

GCTTGTGATAGCACGCCAGAAAGTCAAGGATGCAGAAAAGCAGTATGAAA

110 120 130 140 150

I I I I I

AGGATCCTGATGACTTCAACAAGAGGGCACTGCATGATCGGGAGAGTGTC

160 170 180 190 200

I I I I I

GCAGCTTCAATACAATCAAAAATTGATGAATTGAAGCGCCAACTTGCCGA

210 220 230 240 250

I I I I I

CAGGATTGCAGCAGGGAAGAATATTGGGCAAGACCGGGATCCTACAGGGG

260 270 280 290 300

I I I I I

TAGAGCCGGGTGATCATCTCAAAGAGAGATCAGCACTAAGCTATGGGAAT

310 320 330 340 350

I I I I I

ACACTGGACCTGAATAGTCTTGACATTGATGAACCTACAGGACAGACAGC

360 370 380 390 400

I I I I I

TGATTGGTTGACCATMTTGTCTATCTGACTTCATTTGTGGTCCTGATCA

410 420 430 440 450

I I I I I

TCTTAAAGGCACTGTACATGTTAACAACAAGAGGCAGGCAGACTTCAAAG

460 470 480 490 500 i i i i i

GACAACAAAGGGATGAGGATCAGATTCAAGGATGACAGCTCATATGAGGA 510 520 530 540 550

I I I I I

TGTCAATGGAATCAGAAAACCCAAACATCTGTATGTGTCAATGCCAAACG

560 570 580 590 600

I I I I I

CCCAATCCAGCATGAAGGCTGAAGAGATAACACCTGGAAGATTCCGCACT

610 620 630 640 650

I I I I I

GCAGTATGTGGGCTATATCCTGCACAGATAAAGGCAAGGAACATGGTGAG

660 670 680 690 700

I I I I I

CCCTGTCATGAGTGTAGTTGGGTTΓTTGGCACTGGCAAAAGATTGGACAT

710 720 730 740 750

I I I I I

CTAGAATTGAAGAATGGCTTGGTGCACCCTGCAAGTTTATGGCGGAATCT

760 770 780 790 800

I I I I I

CCAATTGCTGGGAGTTTATCTGGGAATCCCGTGAATCGTAACTATATCAG

810 820 830 840 850

I I I I I

ACAGAGACAAGGTGCACTTGCAGGGATGGAGCCAAAGGAATTTCAAGCCC

860 870 880 890 900

I I I I I

TCAGGCAACATGCAAAGGATGCTGGGTGTACACTGGTTGAGCATΛTTGAG

910 920 930 940 950

I I I I I

TCACCATCATCAATATGGGTGTTTGCTGGGGCCCCTGATAGGTGTCCACC

960 970 980 990 1000

I I I I I

MCATGTTTGTTTGTCGGAGGGATGGCTGAGTTAGGTGCCTTCTTTTCTA

1010 1020 1030 1040 1050

I I I I I

TACTTCAGGATATGAGGAACACAATCATGGCTTCAAAAACTGTGGGCACA 1060 1070 1080 1090 1100

I I I I I

GCTGATGAAAAGCTTCGAAAGAAATCATCATTCTATCAATCATACCTCAG

1110 1120 1130 1140 1150

I I I I I

ACGCACACAATCAATGGGAATACAACTGGACCAGAGGATAATTGTTATGT

1160 1170 1180 1190 1200

1 I I I I

TTATGGTTGCCTGGGGAAAGGAGGCAGTGGACAACTTTCATCTCGGTGAT

1210 1220 1230 1240 1250

I I I I I

GACATGGACCCAGAGCTTCGTAGCCTGGCTCAGATCCTGATTGACCAGAA

1260 1270 1280 1290

I I I I

AGTAAAAGAAATCTCGAACCAGGAACCTTTGAAACTCTAA

2. A recombinant expression plasmid containing the Seoul virus R22 nucleocapsid gene according to claim 1.

3. The recombinant expression plasmid according to claim 2, which is the expression plasmid pET-sNP constructed by cloning the Seoul virus R22 nucleocapsid gene of claim 1 into vector pET-3a.

4. A transformed Escherichia coli with the recombinant expression plasmid according to claim 2.

5. The transformed microorganism according to claim 4, which is the transformed E. coli BL(pET-sNP) produced by introducing the expression plasmid of claim 3 into E. coli BL21(DE3).

6. A fused form of Seoul virus R22 nucleocapsid protein having the following amino acid sequence isolated and purified from culture of the transformed microorganism of claim 4:

10 20 30 40 50

I 1 I I I

MASMTGGQQMGRGSMATMEEIQREISAHEGQLVIARQKVKDAEKQYEKDP

60 70 80 90 100 DDFNKRALHDRESVAASIQSKIDELKRQLADRIAAGKNIGQDRDPTGVEP

110 120 130 140 150

I I I I I

GDHLKERSALSYGNTLDLNSLDIDEPTGQTADWLTI IVYLTSFWLI ILK

160 170 180 190 200

I I I I I ALYMLTTRGRQTSKDNKGMRIRFKDDSSYEDVNGIRKPKHI.YVSMPNAQS

210 220 230 240 250

I I I I I

SMKAEEITPGRFRTAVCGLYPAQIKARNMVSPVMSVVGFLΛLAKDWTSRI

260 270 280 290 300 EEWLGAPCKFMAESPIAGSLSGNPVNRNYIRQRQGALAGMEPKEFQALRQ

310 320 330 340 350

I I I I I

HAKDAGCTLVEHIESPSSI VFAGAPDRCPPTCLFVGGMAELGAFFSILQ

360 370 380 390 400

I I I I I DMRNTIMASKTVGTADEKLRKKSSFYQSYLRRTQSMGIQLDQR11VMFMV

410 420 430 440

I I I I

AWGKEAVDNFHLGDDMDPELRSLAQ IL IDQKVKE I SNQEPLKL

7. A pharmaceutical composition for diagnosis of HFRS comprising the fused form of Seoul virus R22 nucleocapsid protein of claim 6.

8. A vaccine for prevention of HFRS comprising the fused form of Seoul virus R22 nucleocapsid protein of claim 6.