CN105018499A - Anti-HBV (hepatitis B virus) PNA (peptide nucleic acid) and applications thereof - Google Patents
Anti-HBV (hepatitis B virus) PNA (peptide nucleic acid) and applications thereof Download PDFInfo
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Abstract
The invention discloses an anti-HBV (hepatitis B virus) PNA (peptide nucleic acid) and applications thereof and belongs to the field of biotechnologies and material science. The sequence of the anti-HBV PNA is 5'-GCAGAGGTGAA-3'; a cell-penetrating peptide Tat is chelated onto the PNA, and a PNA complex, that is, NH2-GRKKRRQRRRPPGC-MPA-GCAGAGGTGAA-3', which can autonomously penetrate through a cell membrane to enter a cell infected with HBVs is obtained. The PNA or the PNA complex contains 11 basic groups, is better in water solubility and extremely low in toxicity to liver cells, can effectively inhibit HBV replication and infection at the cell level or the animal level and can be used for preparing anti-HBV drugs as well as drugs for treating or preventing HBV infection or diseases caused by HBV infection, and the production method is convenient.
Description
Technical field
The invention belongs to biotechnology and material science, relate to peptide nucleic acid(PNA) of a kind of anti-hepatitis B virus and uses thereof.
Background technology
Hepatitis B virus (HBV) a kind ofly has addicted to liver property, do not cause cytopathic DNA virus.Hepatitis B virus infection is all medical treatment & health problem great in world wide all the time.The current whole world 3.5 ~ 400,000,000 people that still have an appointment are subject to the puzzlement of chronic viral hepatitis B.The persistent infection of hepatitis B virus (HBV) is significantly risen making the probability of patient's suffering from chronic activity hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC).The liver cancers that the whole world exceedes half is all relevant with hepatitis B virus infection.
Current mainly Interferon, rabbit and the nucleic acid analog class medicine being applied to treating hepatitis B clinically.But because Interferon, rabbit easily produces toxic side effect, and the nucleic acid analogs such as such as lamivudine (3TC) easily cause resistance in clinical prolonged application.
In recent years, research and development various new anti-hepatitis B medicine or actives Quality Research are in the news in succession, mostly adopt and choose newtype drug target or reduce the methods such as poisonous side effect of medicine, to promote the performance of medicine.But, still do not have to come out by the medicine that infects of perfect treatment hepatitis B virus (HBV).Design, the novel anti-hbv drug of R and D are still very important and urgent.
Peptide nucleic acid(PNA) (PNA) is a kind of polymer of synthetic, is usually applied to disease treatment with pairing in conjunction with target calibration method or for clinical molecular diagnosis.Peptide nucleic acid(PNA) (PNA) is the nucleic acid analog with synthetic skeleton.But because its skeleton is electric neutrality and snappiness is extremely strong, peptide nucleic acid(PNA) shows and DNA or the very high bonding force of RNA molecule and specificity, and extremely difficult by nuclease and proteolytic enzyme identification and degraded.Therefore there is the longer transformation period.In addition, peptide nucleic acid(PNA) (PNA) molecule often toxicity is very low, has larger application potential.In materialogy, in order to enable a kind of material molecule realize greater functionality, usually peptide nucleic acid(PNA) (PNA) and some other functional groups are carried out chelating.
Summary of the invention
The object of the present invention is to provide a kind of peptide nucleic acid(PNA) of anti-hepatitis B virus.Another object of the present invention is to the peptide nucleic acid(PNA) complex body that a kind of anti-hepatitis B virus is provided.Another object of the present invention is the purposes providing above-mentioned peptide nucleic acid(PNA), peptide nucleic acid(PNA) complex body.
Object of the present invention is achieved through the following technical solutions:
A peptide nucleic acid(PNA) for anti-hepatitis B virus, its nucleotide sequence is 5 '-GCAGAGGTGAA-3 ' (SEQ ID NO.1).
The peptide nucleic acid(PNA) complex body of anti-hepatitis B virus is chelating cell-penetrating peptide on above-mentioned peptide nucleic acid(PNA).Described cell-penetrating peptide is preferably Tat (GRKKRRQRRRPPGC, SEQ ID NO.2), and when cell-penetrating peptide is Tat, the amino acid of this peptide nucleic acid(PNA) complex body and nucleotide sequence are: NH
2-GRKKRRQRRRPPGC-MPA-GCAGAGGTGAA-3 ', MPA are 3-maleimidoproprionic acid group.
Above-mentioned peptide nucleic acid(PNA) or the application of peptide nucleic acid(PNA) complex body in the medicine preparing anti-hepatitis B virus.
Above-mentioned peptide nucleic acid(PNA) or peptide nucleic acid(PNA) complex body are preparing the application treated and/or prevented in the medicine of hepatitis B.
Tool of the present invention has the following advantages and beneficial effect:
(1) peptide nucleic acid(PNA) of the present invention and peptide nucleic acid(PNA) complex body contain 11 bases, and production method is easy, no cytotoxicity, better water-soluble.
(2) peptide nucleic acid(PNA) of the present invention and peptide nucleic acid(PNA) complex body antiviral activity high, can autonomous permeates cell membranes enter by the cell of HBV infection, obvious restraining effect has been copied to HBV, just can 50% be reached to the inhibiting rate of HBV when its concentration is at about 1 μM, it has the function suppressing hepatitis B virus infection, inhibition is remarkable, has no side effect to liver cell.Compared with existing antiviral, peptide nucleic acid(PNA) complex body of the present invention has and more directly and effectively acts on.
Accompanying drawing explanation
Fig. 1 is the Design and synthesis schema of Tat-PNA-DR.
Fig. 2 is the Mass Spectrometric Identification figure of PNA-DR.
Fig. 3 is the result figure of HPLC separation and purification Tat-PNA-DR.
Fig. 4 is the Tat-PNA-DR mass spectroscopy figure after HPLC separation and purification.
Fig. 5 is that different concns Tat-PNA-DR detects column result figure to HepG2.2.15 cytotoxicity.
Fig. 6 is the result figure of different concns Tat-PNA-DR on HBV e antigen concentration impact in HepG2.2.15 cell line supernatant, X-coordinate is the concentration (μM) of Tat-PNA-DR, ordinate zou is the amount of viral protein after the Tat-PNA-DR process of respective concentration, not add the protein content of the experimental group of Tat-PNA-DR (namely Tat-PNA-DR concentration is 0 μM) as 100%.
Fig. 7 is the result figure of different concns Tat-PNA-DR on HBV s antigen concentration impact in HepG2.2.15 cell line supernatant, X-coordinate is the concentration (μM) of Tat-PNA-DR, ordinate zou is the amount of viral protein after the Tat-PNA-DR process of respective concentration, not add the protein content of the experimental group of Tat-PNA-DR (namely Tat-PNA-DR concentration is 0 μM) as 100%.
Fig. 8 is Tat-PNA-DR result figure to HBV e antigen inhibition in HBV chmice acute infection model, and X-coordinate is the grouping of laboratory animal Different treatments, and ordinate zou is the concentration of e antigen in mice serum.
Fig. 9 is Tat-PNA-DR result figure to HBV s antigen inhibition in HBV chmice acute infection model, and X-coordinate is the grouping of laboratory animal Different treatments, and ordinate zou is the concentration of s antigen in mice serum.
Embodiment
Below in conjunction with embodiment, further detailed description is done to the present invention; but embodiments of the present invention are not limited thereto; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
The preparation of embodiment 1 Anti-HBV activity peptide nucleic acid(PNA) complex body Tat-PNA-DR
The Design and synthesis flow process of Anti-HBV activity peptide nucleic acid(PNA) complex body of the present invention is shown in Fig. 1.First search out one section of nucleic acid region DR1 on HBV pregenome RNA, its sequence is: 5 '-UUCACCUCUGC-3 '.According to the nucleotide sequence of DR1, design the peptide nucleic acid(PNA) PNA-DR of complementary pairing with it, PNA-DR sequence is 5 '-MPA-GCAGAGGTGAA-3 '.For the ability making this peptide nucleic acid(PNA) have autonomous permeates cell membranes, again by PNA-DR and cell-penetrating peptide Tat (GRKKRRQRRRPPGC, SEQID NO.2) carry out chelating and namely obtain Anti-HBV activity peptide nucleic acid(PNA) complex body Tat-PNA-DR, its amino acid and nucleotide sequence are: NH
2-GRKKRRQRRRPPGC-MPA-GCAGAGGTGAA-3 ', MPA are 3-maleimidoproprionic acid group.The concrete preparation process of Tat-PNA-DR is as follows:
A: synthesize with the PNA-DR of target sequence DR1 complementation
Use the method synthetic PNA oligopolymer of solid phase synthesis, and with fluorenylmethyloxycarbonyl/hexichol first carbonyl-protection PNA monomer.Addition PNA chain one by one on Rink Amide-AM resin.PNA monomer reacts by addition with the fluorenylmethyloxycarbonyl that goes repeated.When PNA monomer is at methyl-2-pyrrolidone (NMP) solution (4 equivalent PNA monomers; 3.6 equivalent HATU (2-(7-azo benzotriazole)-N; N; N'; N'-tetramethyl-urea phosphofluoric acid ester); 8 equivalent NMM (methylmorpholine)) in coupling time; piperidines dimethyl formamide (DMF) solution (20%; v/v) be used to remove fluorenylmethyloxycarbonyl blocking group; diacetyl oxide in DMF and 2,6-lutidine mixture are used to add cap.After completing in steps, clean resin 4 times, to remove residual reactant with DMF.MPA (3-maleimidoproprionic acid) group is connected to least significant end in the same way.After all linked reactions all complete, with 3 the also vacuum-drying of DMF, methyl alcohol and methylene dichloride cleaning resin.Then, use the mixture (95:3:2, v/v/v) of TFA, meta-cresol and water to process 2 hours, PNA chain is gone protect and dissociate completely, to be separated from resin.By the method for rotary evaporation reaction product to be filtered and concentrated, then with excessive ice ether sedimentation.By pelleting centrifugation and vacuum-drying.Carry out MALDI-TOF Mass Spectrometric Identification with Axima TOF2 mass spectrograph (Shimadzu, Kyoto, Japan) and confirm product correct (Fig. 2).
The chelating of B:PNA-DR and cell-penetrating peptide Tat
Tat polypeptide (GRKKRRQRRRPPGC-OH) is bought from GL Biochem Inc. company (Shanghai, China).At Tat C-terminal design cysteine residues to make it to react with the maleimide base group in PNA sequence, generate Tat-PNA-DR complex body.With the ratio of mol ratio 1:2 MPA-PNA and Tat is dissolved in distilled water and in stable stirred under nitrogen flow 2 days to ensure its anaerobic condition.First this solution carry out dialysis purifying with dialysis tubing (molecular weight 2000).Then, further separation and purification (Fig. 3) is carried out with high performance liquid chromatography (HPLC).Each main peak of HPLC display is collected, and carries out identifying (Fig. 4) with mass spectrum, and that determines required product goes out peak position.Solution collected by correct peak is carried out lyophilize, to obtain the final product Tat-PNA-DR of high purity (purity reaches more than 95%).
The Cytotoxic detection of embodiment 2 peptide nucleic acid(PNA) complex body Tat-PNA-DR
1, material: the cultivation of HepG2.2.15 cell
HepG2.2.15 clone, purchased from China typical culture collection center.
DMEM substratum: 10% calf serum (FCS, GIBCO-BRL), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.
Culture condition: 37 DEG C, 5%CO
2incubator is cultivated.
2, concrete operation method:
MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt) is a kind of dyestuff, and MTT experiment detects the experimental technique of Growth of Cells and survival.Its principle is that succinodehydrogenase in viable cell plastosome can reduce MTT, makes it become hepatic crystallization first a ceremonial jade-ladle, used in libation, and accumulates in cell; The cell of death is then without this function.After adding DMSO, the first a ceremonial jade-ladle, used in libation that can be formed in dissolved cell.Measure the photoabsorption of 570nm with enzyme-linked immunosorbent assay instrument, viable cell quantity can be reflected.Concrete steps are as follows:
(1) collect logarithmic phase HepG2.2.15 cell, spread 96 orifice plates with the certain density cell suspension of DMEM substratum furnishing, every hole adds 180 μ L, and final cell density is about 10
4individual/hole.
(2) then every hole adds the substratum of 20 μ L containing different concns Tat-PNA-DR, the final concentration of Tat-PNA-DR is made to be respectively 100 μMs, 50 μMs, 25 μMs, 12.5 μMs, 6.25 μMs, 3.13 μMs, 1.56 μMs, 0.78 μM and 0 μM, the multiple hole of each concentration 5.After adding Tat-PNA-DR, culture plate is put in cell culture incubator, cultivates 2 days.
(3) add 20 μ L MTT phosphate solutions (5mg/mL, pH 7.4) in every hole, continue 37 DEG C and cultivate termination cultivation after 4 hours.Removed by cell conditioned medium liquid, every hole adds 200 μ L DMSO, and vibration 10min, makes crystallization fully dissolve.
(4) in microplate reader, setting measurement wavelength is 570nm, reference wavelength is 630nm, measure each hole absorbance value, the survival rate of HepG2.2.15 cell during calculating Tat-PNA-DR different concns, draw cell survival rate histogram, the results are shown in Figure 5, show that Tat-PNA-DR still has no side effect to liver cell under up to 100 μMs of concentration.
Embodiment 3 cell levels peptide nucleic acid(PNA) complex body Tat-PNA-DR is to the suppression of hbv replication
1, material:
The cultivation of HepG2.2.15 cell: HepG2.2.15 clone, purchased from China typical culture collection center.DMEM substratum: 10% calf serum (FCS, GIBCO-BRL), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.Culture condition: 37 DEG C, 5%CO
2incubator is cultivated.
ELISA kit: hepatitis B virus e antigen diagnostic kit---vertical readable series, hepatitis B virus surface antigen diagnostic kit---vertical readable series, purchased from Ke Hua biotechnology limited-liability company (Shanghai).
2, concrete operation method:
With peptide nucleic acid(PNA) complex body Tat-PNA-DR process HepG2.2.15 cell, HBeAg and HBsAg in its cell conditioned medium uses ELISA kit to detect.
(1) collect logarithmic phase HepG2.2.15 cell, spread 24 orifice plates with the certain density cell suspension of DMEM substratum furnishing, every hole adds 480 μ L, and final cell density is about 8 × 10
4individual/hole, cultivates 12 hours, treats cell attachment.
(2) then every hole adds the substratum of 20 μ L containing different concns Tat-PNA-DR, makes the final concentration 10 μMs, 1 μM, 0.1 μM and 0 μM respectively of Tat-PNA-DR, the multiple hole of each concentration 3.After adding Tat-PNA-DR, culture plate is put in cell culture incubator, cultivates 2 days.Lamivudine (3TC) is as drug control, and its final concentration is 1 μM.
(3) the Elisa elisa plate that cell culture supernatant sample 50 μ L adds HBeAg antibody or HBsAg antibody bag quilt is got.Then every hole adds 50 μ L enzyme connection mixed solutions, and 37 DEG C of standing lucifuges hatch 30min.Using HepG2 cell conditioned medium as negative control, the positive control that not enzyme-added mixed solution provides as blank and test kit, and use the standard substance in test kit to build typical curve.
(4) hatch rear normal temperature and use 1 × wash liquid elisa plate, leave standstill 60s after adding 1 × washing lotion, dry.Repeated washing 6 times.
(5) in elisa plate, every hole adds each 50 μ L of nitrite ion A and B respectively, and 37 DEG C of standing lucifuges hatch 15min.
(6), after hatching end, in elisa plate, every hole adds 50 μ L reaction terminating liquids.After mixing, the inherent microplate reader of 15min reads absorbance value with 490nm wavelength.
With the OD value drawing standard curve of standard substance, and sample OD value is substituted into typical curve equation, obtain sample virus titre, according to following formulae discovery inhibiting rate, the results are shown in Table 1-2 and Fig. 6-7.
Inhibiting rate=(negative control titre-testing sample titre)/negative control titre × 100%.
Table 1 different concns Tat-PNA-DR is to the suppression result of HBV e antigen
| Tat-PNA-DR(μM) | 10 | 1 | 0.1 | 0 | 3TC |
| E antigen relative quantity (%) | 83.12 | 66.18 | 7.08 | 100 | 98.69 |
Table 2 different concns Tat-PNA-DR is to the suppression result of HBV s antigen
| Tat-PNA-DR(μM) | 10 | 1 | 0.1 | 0 | 3TC |
| S antigen relative quantity (%) | 70.11 | 51.42 | 31.06 | 100 | 100.86 |
The above results shows that the propagation of peptide nucleic acid(PNA) complex body Tat-PNA-DR of the present invention to hepatitis B virus has efficient restraining effect: when the concentration of Tat-PNA-DR is about 1 μM, it can reach 50% to the inhibiting rate of hepatitis B virus e, s antigen.
Embodiment 4 animal horizontal peptide nucleic acid(PNA) complex body Tat-PNA-DR is to the suppression of hbv replication
1, material
Balb/C mouse: purchased from Disease Control and Prevention Center of Hubei Province.
Rearing conditions: SPF level animal rearing center.
2, concrete operation method:
A:BALB/c chmice acute HBV infection model construction and Tat-PNA-DR process
(1) BALB/c mouse used is all male, 6-7 week age, weight is about about 20g.Use BALB/c mouse 28 altogether, be divided into four groups, often organize 7.Be respectively blank group (Blank), HBV infection group, Tat-PNA-DR treatment group and 3TC drug control group.
(2) ultrapure is inserted the genomic PUC18 plasmid (pUC18-HBV1.3plasmid of HBV 1.3 times, Zhao Z, Hong W, Zeng Z, Wu Y, Hu K, Tian X, Li W, Cao Z.Mucroporin-M1inhibits hepatitis B virusreplication by activating the mitogen-activated protein kinase (MAPK) pathway anddown-regulating HNF4alpha in vitro and in vivo [J] .The Journal of biological chemistry 2012; 287 (36): 30181-90.) totally 20 μ g are dissolved in the physiological saline of Mouse Weight 8% volume.Each mouse in HBV infection group, Tat-PNA-DR treatment group and 3TC drug control group all tail vein injection contains the physiological saline of 20 μ g HBV, 1.3 times of geneome plasmids.And Blank group (blank group) is not for intravenous injection equal-volume is containing the physiological saline of plasmid.
(3) second day after injection, Tat-PNA-DR treatment group mouse injected mouse tail vein with the Tat-PNA-DR of 12.5mg/kg (200 μ L).And HBV infection group and blank group inject isopyknic physiological saline.3TC drug control group injection equal-volume 3TC solution, makes final Plasma Concentration be 1 μM.
Within (4) the 3rd days, receive mouse blood and liver specimens.Liver specimens is fixed on 4% paraformaldehyde solution of about 10 times of volumes.Mouse blood uses eyeground blood-collecting method blood sampling.The Mouse whole blood obtained by kapillary puts into 37 DEG C of incubators, incubation 1 hour, then puts into 4 DEG C of refrigerator 15min, makes clot solidification shrinkage, upper serum is transferred to a new 1.5mL centrifuge tube, for subsequent use.
B: HBeAg and HBsAg Detection of antigen in mouse blood
Experiment adopts the ELISA kit (with embodiment 3) of Ke Hua biotechnology limited-liability company (Shanghai), operates by test kit specification sheets.Concrete operations are as follows:
(1) proportional diluted standardized solution, preparation standard substance.
(2) in enzyme plate, every hole adds 40 μ L example reaction liquid, then adds 10 μ L samples respectively, the repetition of three, each sample.And blank is set, negative control, and HepG2.2.15 cell conditioned medium positive control.
(3) after enzyme plate film shrouding 37 DEG C hatch 30min.
(4) plate is washed.Every hole adds washing lotion, leaves standstill 30sec, removes washing lotion, pat dry.Repeat 5 times, and enzyme plate is patted dry.
(5) except blank, every hole adds HRP cross-linking reagent 50 μ L, hatches 30min for 37 DEG C.
(6) again plate is washed.Repeating step (4).
(7) every hole adds 50 μ L nitrite ion A and 50 μ L nitrite ion B respectively, and 37 DEG C of lucifuges hatch 15min.
(8) every hole adds 50 μ L reaction terminating liquid termination reactions.
(9) in microplate reader, arrange reading wavelength is 450nm, and with reference to wavelength 630nm, reading of data, according to antigen concentration in standard substance calculation sample.
The results are shown in Figure 8-9, in Tat-PNA-DR treatment group mouse blood, HBeAg and HBsAg comparatively HBV infection group reduction, illustrates that Tat-PNA-DR significantly can suppress HBeAg and HBsAg in HBV acute infection mouse model blood.3TC drug control group due to the mode of action of 3TC be suppress the generation of HBV gene group, to antigen presentation without remarkable effect.Blank group does not detect antigen, meets expection.
Above-described embodiment result shows, Tat-PNA-DR, to cytotoxic side effect, can suppress copying and infecting of HBV, can be used for preparing Anti-HBV drugs and HBV infection and causes treatment or the prophylactic agent of disease.
Claims (7)
1. a peptide nucleic acid(PNA) for anti-hepatitis B virus, is characterized in that: its nucleotide sequence is 5 '-GCAGAGGTGAA-3 '.
2. a peptide nucleic acid(PNA) complex body for anti-hepatitis B virus, is characterized in that: chelating cell-penetrating peptide on peptide nucleic acid(PNA) according to claim 1.
3. peptide nucleic acid(PNA) complex body according to claim 2, is characterized in that: the aminoacid sequence of described cell-penetrating peptide is GRKKRRQRRRPPGC.
4. peptide nucleic acid(PNA) according to claim 1 is preparing the application in hepatitis B virus resisting medicine.
5. peptide nucleic acid(PNA) according to claim 1 is preparing the application treated and/or prevented in hepatitis B medicament.
6. the peptide nucleic acid(PNA) complex body described in Claims 2 or 3 is preparing the application in hepatitis B virus resisting medicine.
7. the peptide nucleic acid(PNA) complex body described in Claims 2 or 3 is preparing the application treated and/or prevented in hepatitis B medicament.
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Application publication date: 20151104 |