CN1186120A - Recombinative human interferon protein as secretory gene in colibacillus - Google Patents

Abstract

A gene expressed lacUV5 promoter and OmpA signal peptide secretion recombined protein code sequence secretion-type carrier has effectively in colibacillus expressed cellulomomas fimi extracellular glucase, which is secreted outside cell. Said secretion carrier has been used to produce interferon alpha 16 protein and its variant and segment with immune and biologic activities. The present invention describes a method to build up recombined DNA structural body lacUV5par8INF and its derivatives, and a method to produce interferon alpha 16 and its variant protein with said secretion carrier and its variant in transgene biologic body and procaryotic cell, especially in colibacillus strain and/or its culture medium.

Description

At expression in escherichia coli as excretory recombinant human interferon alpha 1 b albumen

The many natural secretory protein of expressing in Bacillus coli cells is degraded or the active inclusion body of formation lifeless matter easily.But, containing the host of secretion vector by employing, they can be secreted in the cell pericentral siphon.Recently, developed expression product has been delivered to excretory system in the supernatant liquor.The aspect that these systems are better than excretory system is 1) reduce to proteolysis minimum and 2) be used for more easily detecting and purification of samples.

Big quantity research adopt the method for secretion vector at the expression in escherichia coli recombinant products.These methods comprise utilization (1) " leakiness " mutant strain (Gikes et al., Bio/Technol., Vol.2, pp259-263.1984); (2) with the solubilising protein (Kobayashi etal., J.Bacteriol., Vol.166, pp728-732,1986) of purpose product coexpression; (3) natural excretory protein or colibacillary outer membrane component and purpose product merge (Nagahari et al., EMBO J., Vol.4, pp3589-3592,1985; Mackman et al., EMBO J.Vol.6, pp2835-2841,1987); (4) strong promoter and efficiently leader sequence with provide effectively expressing and secretion (by unknown mechanism cause the secretion) purpose product (Pages et al., J.Bacteriol, Vol.169, pp 1368-1390,1987; Suominen etal., Gene, Vol.61, pp165-176,1987; Guo et al., FEMS Microbiol.Lett., Vol.49, pp279-283,1988; Wong et al., U.S. Patent No. 5,223,407,1993; Wong et al., European patent No.EP0357291B1,1996).In these methods, last a kind of mode is the simplest.In addition, it uses normal host strain so that can obtain the maximum expression and the maximum cells produce amount of desired protein.In addition, this method can utilize the body endopeptidase from the required product of signal peptide cracking, does not need extra cleavage step just can discharge the purpose product from fusion rotein thus.

Utilize above-mentioned last a kind of method, structure is called Tac box (Wong et al., 1993﹠amp; 1996 above) the synthesis secretion box, produce recombinant protein with secretion in intestinal bacteria.Described Tac box contains necessary all regulatory elements of high level expression protein in intestinal bacteria, the tac promotor that comprises heterozygosis, the lac operon that is used for transcriptional regulatory, total ribosome bind site, omp.A leader sequence and trp A transcription terminator (Wong et al., 1993 and 1996 above).In order to make up secretion vector, the Tac box is cloned among the pUC18, wherein also found to cross the lac I that expresses the Lac repressor ^qThe dividing element par of gene and plasmid pSC101 (Wong et al., 1993﹠amp; 1996 above).

Remove application Tac box intestinal bacteria excretory system and produce various heterologous proteins, comprise hEGF, hPTH, the peptide that IL-6 is relevant with Rat parathyroid hormone 1-34 outer (Wong et al., 1993﹠amp; 1996 above; Rabbani et al., J.Bone Min.Res., Vol.6[replenishes 1], Abstract 133, S116,1991), exist under the IPTG inductive condition, use the Tac box, express come from C.fimi (O ' Neill et al., Gene, Vol.44, pp 325-330, the extracellular dextranase (Exg of cex gene 1986a), use and produce the look substrate, right-nitrophenyl-β-D-cellobiose glycosides (pNPC) can detect expression product easily.), the secretion production of expression product will cause cell rapidly dead (Fig. 1), however under the same conditions, when not having IPTG, the cell of expressing Exg is with normal speed growth (Fig. 1).Will Tac box secretion vector (above-mentioned) and cex gene (O ' Neill et al., the recombinant precursor that forms between above) is called tacIQpar8cex (Fig. 2).

Exg (the Uml that non-inductive and inductive JM101 (tacIQpar8cex) express ^-1) there is very big difference (Fig. 1 and table 1) the overall yield aspect, this may be that difference (Fig. 3) owing to viable count between two kinds of cultures causes, still, and two kinds of culture excretory Exg activity (Uml ^-1) very similar (table 1).The result shows, two kinds of cultures are effective production extracellular Exg all.

The tacIQpar8cex construct is under non-inductive condition, can be in intestinal bacteria high level expression Exg, but the necrocytosis that causes behind abduction delivering Exg shows that with the result that can not effectively secrete Exg (table 1) it produces other exogenous protein for secretion may not be best.Therefore, perhaps can be used to improve the expression justacrine of Exg and other recombinant proteins better to the extracellular than the promotor a little less than the tac.Select lac (Hawley et al., Nucleic Acids Res., Vol.11, pp 2237-2255,1983) and lacUV5 promotor (Russell et al., Gene, Vol.20, pp.231-243,1982 for this reason; Silverstoneet al, Proc.Natl.Acad.Sci.U.SA, Vol.66, pp773-779,1970) check above-mentioned hypothesis.Utilize point mutation (Kunkel, Proc.Natl.Acad.Sci.U.S.A., Vol.82, pp488-492,1985) technology, make up two tacIQpar8cex derivatives and be used for research, be referred to as lacIQpar8cex (Fig. 4) and lacUV5par8cex (Fig. 5), they contain lac and lacUV5 promotor respectively.

(Fig. 1) compare with JM101 (tacIQpar8cex), JM101 (lacIQpar8cex) (Fig. 6) and JMI01 (lacUV5par8cex) inducing culture thing (Fig. 7) improvement is all arranged aspect viability.When the efficient of the lacIQpar8cex of evaluation expression Exg and lacUV5par8cex construct, the culture of two kinds of constructs has only been expressed low-level Exg activity (table 1).Data show, the lacI that these constructs are expressed ^qThe repressor of gene is being regulated lac and lacUV5 promotor scrupulously.But check in case induce to have removed, have only the culture of JM101 (lacUV5par8cex) that tangible increase is being arranged aspect the Exg expression, then not the having of JM101 (lacIQpar8cex) (table 1) with IPTG.Not only in cell in the sample, and in the culture supernatants sample, also detect the increase (table 1) of expression product.Although two kinds of total Exg activity (Uml that culture is expressed ^-1) similar level (table 1) arranged, the specific activity of JM101 (lacUV5par8cex) inducing culture thing secretion Exg is inductive JM101 (tacIQpar8cex) culture active high 4 times not.Therefore, can be by inducing the mode of JM101 (lacUV5par8cex) expression-secretion Exg, the research optimal growth condition is to obtain expressing justacrine to extracellular protein optimum yields.

The ability (table 1 and Fig. 8) that the lacUV5par8cex construct is expressed high-level extracellular Exg has shown that it arrives extracellular potential use at other heterologous proteins of expression in escherichia coli such as interferon alpha 1b justacrine.By replacing the cex gene order, with encoding sequence (Wu etal., the Fundamental﹠amp of human interferon alpha 1 b gene; Clinical Trials of Recombinant Drugs, People ' s HealthPress, p.155,1996) be cloned in (Fig. 9) in the lacUV5par8cex construct.After clone and the mutagenesis operation, in final secretion construct, interferon alpha 1b gene and ompA leader sequence are merged exactly, be called lacUV5par8INF (Figure 10).Determined that with immunology and biological test (table 2) construct with lacUV5par8INF (is called pINF, Fig. 9) has successfully expressed interferon alpha 1b protein.

According to the present invention, we provide recombinant DNA construction body lacUV5par8INF and derivative thereof, and the preserving number of recombinant DNA construction body lacUV5par8INF is that the described recombinant DNA construction body of CCTCC M96024. and derivative thereof are at prokaryotic cell prokaryocyte, particularly express interferon alpha 1b protein and/or have interferon alpha 1b protein immunocompetence and/or the proteinic varient of bioactive interferon alpha 1b in coli strain.Interferon, rabbit and/or its varient of being expressed by cell transformed are secreted in the substratum, can detect it with immunological method and/or biological method test.Can also use standard chromatographic technique purifying alpha-interferon and/or its varient, adopt polyacrylamide gel electrophoresis (PAGE) to analyze the product of purifying then.

According to the present invention, we provide the construction process of recombinant DNA construction body lacUV5par8INF, this method is by the proteinic gene order subclone of the plain α 1b of coded interference is made up in the lacUV5par8cex excretion vector, and described recombinant DNA construction body can be expressed interferon alpha 1b active protein justacrine to the extracellular.

According to the present invention, we provide the construction process of the derivative of recombinant DNA construction body lacUV5par8INF, this method is to form in the lacUV5par8cex excretion vector by mutant or fragment subclone with interferon alpha 1b gene, and its varient justacrine that can express interferon alpha 1b active protein is to the extracellular.With regard to immunocompetence and/or biological activity, this varient is equal to natural interferon alpha 1b protein on function.

According to the present invention, we provide the construction process of the derivative of recombinant DNA construction body lacUV5par8INF, this method is by will forming in interferon alpha 1b gene or its mutant subclone lacUV5par8cex recombinant DNA construction body after modify, and the modification of lacUV5par8cex is to adopt promotor, ribosome binding sequence, opmA leader sequence, transcription terminator, lacI commonly used in the prokaryotic organism ^qGene and/or par element replace the lacUV5 promotor among the lacUV5par8cex, total ribosome binding sequence, opmA leader sequence, trpA transcription terminator, lacI ^qGene and/or par element carry out, and described derivative all can be expressed active protein or its varient of interferon alpha 1b, for example adopt tac or lac promotor to replace the lacUV5 promotor, perhaps replace amicillin resistance etc. with tetracyclin resistance.

The present invention also comprises any mutant that comes from lacUV5par8INF, and (as changing its amicillin resistance is other selective markers to come from the lacUV5par8INF isomer, tetracyclin resistance mark for example) any mutant is expressed interferon alpha 1b protein or its varient.

Also the lacUV5par8INF or derivatives thereof can be changed over to active protein or its varient of expressing interferon alpha 1b in the transgenic organism according to the present invention.

With reference now to following accompanying drawing, illustrates the present invention.

Fig. 1 illustrates that the tacIQpar8cex construct (as shown in Figure 2) that makes up with the Tac box is in intestinal bacteria JM101 bacterial strain, through (■-) or without extracellular Exg activity behind (-) IPTG abduction delivering.The process of inducing is that in 2X YT substratum, 30 ℃ of about 200 RPM shaking cultured cells are at A ₅₅₀Value is about at 0.3 o'clock, adds the final concentration that IPTG reaches 0.1mM.Listed data are the average ± SEM values from different tests (n=3).

Fig. 2 is a diagram of expressing the tacIQpar8cex construct of Exg.Indicate the encoding sequence of ripe Exg (cex), crossed the gene (lacI that expresses the Lac repressor ^q), from the dividing element (par) of pSC101, and amicillin resistance (Amp ^R) the bla gene.Indicated the element of Tac box: tacP=tac promotor, lacO=lac operon, RBS=ribosome bind site, ompA=ompA leader sequence, the series connection terminator codon of STOP=in three reading frames, term=transcription terminator.Represent the non-coding region of cex gene and the direction of genetic expression with shade rod and arrow respectively.Fig. 3 (A) expression through or without the viable count of IPTG inductive intestinal bacteria JM101 (tacIQpar8cex).Inductive condition is identical with description in Fig. 1 explanation.Bacterial count is at 2X YT plate and contain on the 2X YT agar plate of penbritin and finish.(----) indicated the viable count at 2X YT agar plate without the intestinal bacteria JM101 (tacIQpar8cex) of IPTG inducing culture.(-) indicated that intestinal bacteria JM101 (tacIQpar8cex) without the IPTG inducing culture is in the viable count that contains penbritin 2X YT agar plate.(--■--) indicated the viable count at 2X YT agar plate through the intestinal bacteria JMI01 of IPTG inducing culture (tacIQpar8cex), and (■-) indicated through the intestinal bacteria JM101 of IPTG inducing culture (tacIQpar8cex) in the viable count that contains penbritin 2X YT agar plate.The enlarged view to the host cell lethal effect is induced in Fig. 3 (B) expression.

Fig. 4 illustrates the lacIQpar8cex construct of expressing Exg.All identical to what describe in the tacIQpar8cex construct explanation at all elements shown in this construct with Fig. 2, remove with lac promotor (lacP) the replacement tac promotor.

Fig. 5 illustrates the lacUV5par8cex construct of expressing Exg.All elements shown in this construct all with Fig. 2 describe in to tacIQpar8cex construct note identical, except that replacing the tac promotor with lacUV5 promotor (lacUV5P).

Fig. 6 represent through or without the viable count of IPTG inductive intestinal bacteria JM101 (lacIQpar8cex).Inductive condition is identical with description in Fig. 1 explanation.Bacterial count is at 2X YT plate and contain on the 2X YT agar plate of penbritin and finish.(----) indicated the viable count at 2X YT agar plate without the intestinal bacteria JM101 (lacIQpar8cex) of IPTG inducing culture.(-) indicated that intestinal bacteria JM101 (lacIQpar8cex) without the IPTG inducing culture is in the plate viable count that contains penbritin 2X YT agar.(--■--) indicated the viable count at 2X YT agar plate through the intestinal bacteria JM101 of IPTG inducing culture (lacIQpar8cex), and (■-) indicated through the intestinal bacteria JM101 of IPTG inducing culture (lacIQpar8cex) in the viable count that contains penbritin 2X YT agar plate.

Fig. 7 represent through or without the viable count of IPTG inductive intestinal bacteria JM101 (lacUV5par8cex).Inductive condition is except at A ₅₅₀Value is about at 1.2 o'clock, adds outside the IPTG, and other are identical with description in Fig. 1 illustrates all.Bacterial count is at 2X YT plate and contain on the 2X YT agar plate of penbritin and finish.(----) indicated the viable count at 2X YT agar plate without the intestinal bacteria JM101 (lacUV5par8cex) of IPTG inducing culture.(-) indicated that intestinal bacteria JM101 (lacUV5par8cex) without the IPTG inducing culture is in the viable count that contains penbritin 2X YT agar plate.(--■--) indicated the viable count at the 2XYT agar plate through the intestinal bacteria JM101 of IPTG inducing culture (lacUV5par8cex), and (■-) indicated through the intestinal bacteria JM101 of the intestinal bacteria inducing culture of IPTG inducing culture (lacUV5par8cex) in the viable count that contains penbritin 2X YT agar plate.

Fig. 8 is illustrated in through (■-) or in the intestinal bacteria JM101 bacterial strain without (-) IPTG inducing culture, the extracellular Exg activity of being expressed by lacUV5par8cex construct (as shown in Figure 5).In the note of Fig. 7, inductive condition is arranged.Listed data are the mean value ± SEM from different tests (n=3).

Fig. 9 illustrates the building process of expressing the proteinic reorganization of interferon alpha 1b pINF construct.

Figure 10 illustrates the building process of expressing the proteinic lacUV5par8INF construct of interferon alpha 1b.Embodiment 1 material and routine techniques (a) coli strain and substratum

Use coli strain JM101 (Yanisch et al., Gene, Vol.33, pp 103-119,1985) and BW313 (Kunkel, together above) to express and propagation plasmid and phage respectively, and be used for the point mutation of dna molecular.Buy intestinal bacteria BHM 71-18 mutS (Δ [lac-proAB], thi, supE, mutS215 ∷ Tn10 from Promega; F ' [lacI ^q, lacZ Δ M15, proAB ⁺]), be used for two strands (ds) DNA mutagenesis by supplier's explanation.

By aforementioned (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2ndedition, Cold Spring Harbour Laboratotry Press, N.Y., 1989) preparation is used to cultivate and keep the 2X YT and the M9 substratum of coli strain.(MUC Sigma) reaches the final concentration of 0.1mM, is used for detecting from bacterium colony excretory Exg activity to add 4-hydroxymethylcoumarin β-D-fiber two pyranoses in the substratum.With containing 70 μ gml ^-1The performance Amp that ampicillin medium screening plasmid transforms ^RThe cell of phenotype and survivaling cell are used for time course research.(b) plasmid and phage construct

The cex gene of the Exg of encoding mature C.fimi can be from plasmid pBR325, obtains in the 1.8-kb Eco RI-HindIII fragment in the derivative of p510.507 (Curry etal., ApplEnviron.Microbiol., Vol.54, pp 476-484).Plasmid lacIQpar8 (Wong et al., 1993﹠amp; 1996, the same) and isomer (Hau, Molecular Virology, the pp 635-636 of pBVX α 1b[pBV867, Ke Yun PublicationCo., 1990)] be used for making up the cex expression of gene and secretion vector is mentioned at relevant report with the gene that interferon alpha 1b is provided.By Tac box (Wong et al., 1993 ﹠amp that will modify; 1996, with above) (wherein lacking the tac promotor) subclone is to the EcoRI of M13mp18 phage vector (Yanisch et al. is same above) and HindIII site and carrier construction RH1 (result who does not deliver).This phage construct helps the mutagenesis of dna molecular.(c) DNA operation

Adopt ordinary method (Sambrook et al., with above) to finish recombinant DNA technology, comprise restriction enzyme digestion, dna modification, cell transformation and transfection, conventional and low-melting agarose gel electrophoresis.The point mutation of single strand dna is carried out (Kunkel, together above) by the method for having delivered.Except the DNA that replaces with uridylic as the template, the sudden change of double-stranded DNA is finished according to some disappearance (USE) method of Deng and Nickoloff (Anal.Biochem.Vol.200, pp81-88,1992) basically.With (the 0.5ugml that contains uridine ^-1) the intestinal bacteria BW313 bacterial strain of 2X YT culture medium culturing prepares the DNA that uridine replaces, then by CsCl-gradient centrifugation (Sambrook et al., with above) purifying.Determine the sudden change of gained by dideoxy sequencing method (Sanger et al., Proc.Natl.Acad.Sci.U.S.A., Vol.74, pp5463-5467,1977).The plasmid of embodiment 2 construction recombination plasmids (a) construction expression c.fimi Exg

The tacIQpar8cex of plasmid lacUV5par8cex (Fig. 1) construction process is as follows:

With EcoRI and PstI digested plasmid p510.507, obtain containing the 0.7kb fragment of cex gene 5 ' end region, mend flat its end with the T4 archaeal dna polymerase.With this fragment cloning to the EcoRV site of RH1 so that 5 ' part of cex gene is placed on the downstream of ompA leader sequence.This product is called intermediate compound I, since cex gene and ompA leader sequence the fusion out of true, use 50bp mutagenic primer OMPAEXO (5 '-CGGCG GCCTC CTTGA GCGTG GTCGC GGCCT GCGCT ACGGT AGCGAAACCA-3 ') suddenlys change to it.Preceding 25 base complementrities of preceding 25 bases of described primer 5 ' end and the cex genes encoding chain of encoding mature Exg product, and back 25 bases annealing of back 25 bases of described primer 3 ' end and ompA leader sequence coding strand.In intermediate product I construct between two annealing fragments not complementary sequence in mutagenic processes, remove, merge accurately obtaining between cex and ompA sequence.The product of gained is called intermediate product II, uses the PstI complete digestion, partly digests to obtain the 0.7-kb fragment with NmI, connects with carrier lacIQpar8 (with identical enzymic digestion) then, to form next intermediate product, tacIQpar8cex '.In order to rebuild the complete encoding sequence of cex, isolating bigger XmnI-PstI fragment is connected with the less XmnI-PstI fragment (1.5-kb) of the p510.507 plasmid purification of part digestion from the tacIQpar8cex ' construct of part digestion.To be called tacIQpar8cex with the construct that cex gene among the lacIQpar8 is rebuild fully.

With USE method (Deng et al., together above), replace in the tacIQpar8cex construct because the promotor of cex transcriptional expression.Contain lac (Hawley et al., with above) or lacUV5 promotor (Russell et al., mutagenesis derivative together above) is called lacIQpar8cex and lacUV5par8cex, obtains as primer with oligonucleotide NATACLAC (5 '-CCGCA TACTT ACTCC CCATA CCCCT GTTTA CACTTTATGC TTCCG GCTCG TATGT TGTGT GGAAT TGTGA GCGGA TAACAATTC-3 ') or oligonucleotide TACUV5 (5 '-GCATA CTTAC TCCCC ATCCCCCTGT TTACA CTTTA TGCTT CCGGC TCGTA TAATG TGTGG AATTGTG-3 ').Six aggressiveness of line refer to-35 and-10 districts of promotor in these primers.(b) plasmid of construction expression interferon alpha 1b

To contain interferon alpha 1b gene plasmid pBVXa1b (Hau, EcoRI-PstI fragment together above), be cloned into EcoRV and add in the RH1 carrier (embodiment 2a) of PstI digestion to form recombinant precursor RH1INF, wherein interferon alpha 1b gene is at Tac box (Wong et al., 1993﹠amp; 1996, with above) in merge with the ompA leader sequence, in clone's process of separating two genetic elements, form the sequence of 7 unnecessary codons.Then with ompA-interferon alpha 1b gene fusion as NruI-PstI fragment subclone in the lacUV5par8cex construct (Fig. 5) of using two kinds of identical enzymic digestions with construct pINF (Fig. 9) in the middle of forming, this construct can be expressed interferon alpha 1b analogue, contains the amino acid of 7 external sources at the N-end of the mature protein of this analogue of escherichia coli expression.

In order to form the final construct of expressing interferon alpha 1b, lacUV5par8INF, as mutagenic primer, at first lack 7 codons that in the RH1INF construct, separate ompA leader sequence and interferon gene sequence with oligonucleotide INF (5 '-ATCCA GGCTG TGGGT CTCAG GGAGA TCACA GGCCT GTGCTACGGT AGCGA AACCA GCCAG-3 ').Cut the mutagenesis product with NruI and PstI enzyme, described product contains the interferon gene that accurately merges with the ompA sequence, to contain then fragment subclone that the ompA-interferon gene merges in the lacUV5par8cex construct (Fig. 5) of using two kinds of identical enzymic digestions to form final construct lacUV5par8INF (Figure 10), think that this construct can be at the ripe interferon alpha 1b protein of expression in escherichia coli through processing.Embodiment 3 detects recombinant products (a) Exg excretory and continues research and active the detection

In each research, be used for bacterium colony from fresh conversion as inoculum.At the substratum of 30 ℃ of insulation inoculations, reach required A with about 200RPM vibration ₅₅₀Value (note of table 1 and accompanying drawing).With the culture separated into two parts.A part (inductive culture) adds the final concentration that IPTG reaches 0.1mM, and another part (not inductive) is not induced.With two kinds of cultures all by aforementioned heat insulating culture, with the sampling carrying out of two hours intervals active detection, viable count and A ₅₅₀Turbidimetry.In order to prepare cell lysates, phosphate buffered saline buffer (Sambrook et al. with half volume, together above) with the cell precipitation washed twice, be resuspended in then in the same buffer of a volume, pressure with 16000psi passes through Frenchpressure mini-cell (SLM Instruments Inc at last, IL., USA) 3 times with its dissolving.

Prior art has been described (Curry et al., Appl.Environ.Microbiol.Vol.54, pp 476-484,1988) and has been used for the active MUC plate test of fluoroscopic examination Exg (MUCase) method.Following detection Exg is to pNPC (Gilkes et al., J.Biol.Chem., Vol.250, hydrolytic activity pp10455-10459) (pNPCase).Usually, (the 100mM sodium phosphate, pH7.0) pNPC with 100 μ l25mM is added in the sample of the suitable dilution of 100 μ l with 20 μ l test damping fluid.Described mixture 37 ℃ of insulations 30 minutes, is passed through to add 1 milliliter of 100mM sodium carbonate solution stopped reaction then.Measure the absorption value of final product at 407nm.One pNPCase of unit per minute produce 1nmol right-nitrophenols.Method (Anal.Biochem., Vol.72, pp248-254,1976) with Bradford is determined protein concn.(b) immunology of interferon alpha 1b and biological test

Use two antibody sandwich methods of the monoclonal antibody of two kinds of anti-interferon alpha 1b according to prior art described (Zhang et al., Bioproducts J.China, Vol.7, pp73-75,1994), recombinantinterferon 1b is carried out immunology detection.In brief, at first use 100 μ l coated antibodies (with the concentration of 20 μ g/ml) to cover one 96 hole flat boards, then 37 ℃ of insulations 2 hours.After removing antibody, be used in 0.05% tween in the Tris damping fluid (pH7.4) with the hole washed twice.Then each culture supernatants sample is added in two holes in duplicate, described flat board is incubated 30 minutes at 37 ℃.With 0.05% tween damping fluid the hole is washed three times, every hole adds horseradish peroxidase bonded antibody, 37 ℃ of insulations 30 minutes, every hole is with tween damping fluid washing 3 times, adds the substrate mixture (be used in the citrate buffer solution with 0.5 μ g/ml prepared at concentrations OPD substrate solution with 1000 times of 30% hydrogen peroxide dilutions) of a prepared fresh then in every hole.Come terminating method by in each hole, adding a 2M sulfuric acid.Measure the absorption value of final reacting mixture at 470nm.The concentration of Interferon, rabbit in the sample is determined in reference with the typical curve of the interferon alpha 1b preparation of purifying.

The ability of resisting virus attack with reference to the Interferon, rabbit of prior art described (Personal communication from Shanghai BioproductResearch Institute) protection WISH cell is determined the biological activity of interferon alpha 1b.The biological activity of one unit Interferon, rabbit is defined as maximum dilution 1ml Interferon, rabbit sample can protect 50% WISH cell to avoid that poison is due to illness attacked and the pathology (Personalcommunication from Shanghai Bioproduct Research Institute) that causes.A makes up tacIQpar8cex as a result

With Tac box (Wong et al., 1993﹠amp; 1996, with above) secrete effectively that various relatively little (＜22kD) heterologous protein prompting, it can promote the secretion than larger protein such as 49-kD C.fimi Exg (O'Neill et al., 1986a is with above).N-end with regard to this enzyme is necessary (the Warren et al. of catalytic activity, proteins, Vol.1, pp335-341,1986) true, determine encoding sequence with Exg, cex (O ' Neill et al., 1986a, together above) in framework, merge, and in the Tac box, be right after after the ompA sequence, wish that from fusion rotein accurately cracking is fallen the OmpA signal peptide to reach active secretion Exg product.With one of secretion construct, lacIQpar8 (Wong et al., 1993﹠amp; 1996, together above) as vector expression cex gene.A series of about subclone and site-directed mutagenesis operation (embodiment 2a) after, the cex gene is accurately merged to form secretion plasmid, tacIQpar8cex (Fig. 2) with ompA in ltcIQpar8.B expresses justacrine Exg by the taclIQpar8cex construct

When the JM101 (tacIQpar8cex) of Exg is expressed in screening, the MUC plate under the UV radiation, the fluorescence halo of periphery of bacterial colonies exist (unlisted data) to show successfully to have expressed Exg.To collect at interval JM101 (tacIQpar8cex) inductive (using IPTG) and inductive (without IPTG) culture not in two hours, the distribution of Exg in the sample is analyzed.Although IPTG of no use induces, the Tac box has still been expressed the cex gene very doughtily in JM101.Be determined at the non-Exg activity (table 1) of inducing sample of time point collection in 12 hours, in this stage, described culture is in back logarithmic phase (data are unlisted).Enzyme level ratio use other system (Curry et al., together above; O ' Neill et al., Appl.Environ.Microbiol., Vol 52, and pp737-743, the high 2-11 of the level of former report 1986b) are doubly.In addition, all detect significant activity level (Fig. 1) in inductive and non-inductive supernatant samples, this has shown that OmpA can guide to Exg in the pericentral siphon, and is secreted in the supernatant liquor from pericentral siphon.

Owing in Tac box construct, have lacI ^qGene element (Fig. 2) will be so when not having IPTG to induce, the expression in the JM101 bacterial strain of heterologous protein will be subjected to strict check (undocumented data).Such adjusting can induce high density cultures to express recombinant protein high-levelly.Notice unexpectedly when even not inducing that JM101 (tacIQpar8cex) also expresses very strong Exg, and the gross activity that the inductive culture is expressed is than its counterpart that " checks " much lower (table 1).Described result shows that it may be deleterious that crossing of Exg expressed for Bacillus coli cells.In addition, a large amount of Exg that produce exist with the dissolved form.

Although the overall yield (Uml of the Exg that from non-inductive and inductive JM101 (tacIQpar8cex), expresses ^-1) be very different, but both are excretory Exg activity (Uml ^-1) very similar (table 1).This shows all production extracellular Exg effectively of the two culture.C IPTG induces the deleterious effect to JM101 (tacIQpar8cex) cell viability

In order to study the restraining effect of Exg, in time course research (Fig. 1), express the viability (Fig. 3) of monitoring bacterial strain simultaneously with Exg to JM101 (tacIQpar8cex) growth.Although it is dead rapidly that described culture normal growth under non-inductive condition, the adding of IPTG cause cell, as cell can not in culture dish, grow (Fig. 3) indicated.When having IPTG, not every cell is all dead immediately, and those can not keep survival (Fig. 3 B) by complete inductive.These cells with may only divide several generations, and those cells that can't register of cell counting early stage (preceding 8 hours) behind induced growth together, might cause the active remarkable increase of excretory Exg (Fig. 1).The active slight increase of detected Exg may be that expression (Fig. 3 B) owing to a small amount of viable cell causes that described cell may be part inductive cell or the cell that only contains a small amount of tacIQpar8cex construct copy in (Fig. 1) at later stages.D utilizes more weak promotor to strengthen Exg and expresses

Although the tacIQpar8cex construct can be at expression in escherichia coli high level Exg, effective secretion (table 1) that it can not provide controllable Exg expression pattern and help described enzyme makes it be unsuitable for secretion and produces Exg.The lethal effect explanation that Exg expresses can be used the production that strengthens extracellular Exg than the promotor a little less than the tac better.Used two kinds of promotor lac (Hawley et al., together above) and lacUV5 (Russell et al., together above) under study for action, the latter is that the former is to insensitive mutant of degrading.

Utilize point mutation technology (Kunkel, with above) and be used for mutagenesis double-stranded DNA branch in USE (Deng et al., together above), by using NTACLAC and TACUV5 (embodiment 2a) to make up two derivatives of tacIQpar8cex respectively, be called lacIQpar8cex and lacUV5par8cex.Two constructs are that the promotor of lac and lacUV5par8cex is the lacUV5 except that the promotor in lacIQpar8cex, and are all identical with tacIQpar8cex.The increment study of E JM101 (lacIQpar8cex) and JM101 (lacUV5par8cex)

The weak promoter lacIQpar8cex and the lacUV5par8cex that express the cex gene in two novel constructs can make host's culture keep normal growth when not having IPTG, also can make it keep good growth (Tu6 ﹠amp when inductor exists; 7).The inducing culture thing of JM101 (lacIQpar8cex) and JM101 (lacUV5par8cex) (Fig. 3) is compared with JM101 (tacIQpar8cex), and the viability of cell improves.But, inducing still of lacUV5par8cex construct has some lethal effects (Fig. 7) to its host, seems pair cell without any deleterious effects (Fig. 6) to adopting inefficient lac promotor to express Exg in JM101 (lacIQpar8cex).

Although the counting of the viable cell that obtains in inductive JM101 (lacIQpar8cex) culture non-ly induces corresponding substrate to lack than it, at all somes detection time, actual cell counting is quite high (Fig. 7).Another point it should be noted that, the wonderful rectification of the plasmid of finding in inductive JM101 (tacIQpar8cex) culture (Fig. 3) is not observed in (Fig. 4) at JM101 (lacUV5par8cex).F improves the production of extracellular Exg with construct lacUV5par8cex

Estimate lacIQpar8cex and the lacUV5par8cex construct efficient when expressing Exg.As indicated by the result who obtains from JM101 (lacUV5par8cex) culture (Fig. 8 and table 1), when not having IPTG to induce, the culture of two kinds of constructs is only expressed low-level Exg activity.Data show that lac and lacUV5 promotor are all tightly by the lacI at these construct coexpressions ^qThe repressor of gene suppresses.But in case induce except that after derepressing by IPTG, the Exg of culture (lacUV5par8cex) (Fig. 8 and table 1) rather than JM101 (lacIQpar8cex) (table 1) production is significantly improved.Not only in cell, detect this raising in the sample, and more obvious in culture supernatants (Fig. 8 and table 1).Inductive JM101 (lacUV5par8cex) excretory Exg activity is four times more than of non-inductive JM101 (tacIQpar8cex) culture (Fig. 1), although total two kinds of Exg activity (Uml that culture is expressed ^-1) level similar (table 1).

In whole time course research (Fig. 3), all in non-inductive JM101 (tacIQpar8cex), record highdensity viable cell, may be able to explain from the tac promoter expression that not exclusively checks, the high-caliber total Exg of (table 1) of detection.If but the relatively activity specific of Exg (serves as that the basis obtains with protein wt (table 1)) or cell counting very clearly is that (particularly in supernatant samples) latter's culture more has productivity than the former.Even can from JM101 (lacUV5par8cex) culture of inducing (Fig. 8) through prolongation IPTG, obtain high-caliber Exg.G expresses justacrine interferon alpha 1b with the pINF construct

Use pINF construct (embodiment 2b and Fig. 9), (embodiment 3b) immunology that usefulness was described in the past and biological method are determined the research at the expression in escherichia coli Interferon, rabbit.Determine at A ₅₅₀=0.5 cell density induces in the supernatant samples of JM101 (pINF) of 12 hours and 14 hours whether have interferon activity with 1mM IPTG.Although construct pINF may only be created in the analogue (antigenicity and biological activity than real Interferon, rabbit are low) that constant protein N-end contains the interferon alpha 1b of 7 additional amino acids, single result from two kinds of tests shows existence immunity and biologic activity (table 2) in supernatant samples.In addition, our result shows that for the first time interferon alpha 1b can be at the expression in escherichia coli justacrine to the extracellular.The different recombinant precursors of table one are expressed the activity of Exg

Than (the U milligram albumen of living ^-1) active (U milliliter ^-1) interior culture medium supernatant tacIQpar8cex-289.3 494.5 561.2 65.5 89.5 10.5tacIQpar8cex of the active percentage restructuring construct interior culture medium supernatant cell of the interior culture medium supernatant cell of inducer cell+5.0 222.5 2.6 58.2 4.3 95.7lacIQpar8cex-22.0,23.5 9.6 0.8 92.3 7.7lacIQpar8cex+55.2 90.2 31.3 3.4 90.2 9.8lacUVpar8cex-110.9,63.3 77.3 1.8 97.7 2.3lacUVpar8cex+403.1 1045.4 273.9 248.1 52.5 47.5

The JM101 bacterial strain that contains different recombinant precursors grows into A550 and after adding or not adding the IPTG inductor that final concentration is 0.1mM, continues to cultivate the activity of measuring Exg after 12 hours at 0.3 o'clock in containing the 2X YT substratum of penbritin.The active sample IPTG induction of immunity activity (ugml of Interferon, rabbit in table two JM101 (pINF) the culture supernatant ^-1) biological activity (IUml ^-1) 1 12 hours 0.020 194 2 24 hour 0.021 162

Claims (11)

1. recombinant DNA construction body pINF, lacUV5par8INF, and derivative, the preserving number of described recombinant DNA construction body pINF is CCTCC M96024

2. the construction process of the described recombinant DNA construction body of claim 1 lacUV5par8INF, this method is by the proteinic gene order subclone of the plain α 1b of coded interference is made up in the lacUV5par8cex excretion vector, and described recombinant DNA construction body can be expressed interferon alpha 1b active protein justacrine to the extracellular.

3. the construction process of the derivative of recombinant DNA construction body lacUV5par8INF in the claim 1, this method is to form in the lacUV5par8cex excretion vector by mutant or fragment subclone with interferon alpha 1b gene, and its varient justacrine that can express interferon alpha 1b active protein is to the extracellular.

4. the construction process of the derivative of recombinant DNA construction body lacUV5par8INF in the claim 1, this method is by will forming in interferon alpha 1b gene or its mutant subclone lacUV5par8cex recombinant DNA construction body after modify, and the modification of lacUV5par8cex is to adopt promotor, ribosome binding sequence, ompA leader sequence, transcription terminator, lacI commonly used in the prokaryotic organism ^qGene and/or par element replace the lacUV5 promotor among the lacUV5par8cex, total ribosome binding sequence, ompA leader sequence, trpA transcription terminator, lacI ^qGene and/or par element carry out, and described derivative all can be expressed active protein or its varient of interferon alpha 1b, for example adopt tac or lac promotor to replace the lacUV5 promotor, perhaps replace amicillin resistance etc. with tetracyclin resistance.

5. contain the host cell that right requires arbitrary recombinant DNA molecules in 1.

6. contain the transgenic organism that right requires arbitrary recombinant DNA molecules in 1.

7. the host cell in the claim 5 is prokaryotic organism.

8. the host cell in the claim 5 is a coli strain.

9. in the organism of claim 5, produce the method for interferon alpha 1b.

10. in the substratum of the organism of claim 5, produce the method for interferon alpha 1b.

11. in the organism of claim 6, produce the method for interferon alpha 1b.

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