CN104862287A - Preparation method of genetic engineering recombinant combined vaccine against hepatitis A and hepatitis E - Google Patents
Preparation method of genetic engineering recombinant combined vaccine against hepatitis A and hepatitis E Download PDFInfo
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Abstract
The invention discloses a preparation method of a genetic engineering recombinant combined vaccine for hepatitis A and hepatitis E and belongs to a method for preparing a vaccine through genetic engineering. According to the method, an HAV (hepatitis A virus) is subjected to reverse transcription, full-length cDNA (complementary deoxyribonucleic acid) is obtained and inserted into a transcription vector carrying a T7 promoter, LFSQA coded sequences of 3 glycine peptide chains, multiple cloning sites and additional 3C protease cutting site peptide chains are built sequentially in a 2A-2B joining region, inserted auxiliary sequences are ggtggcgga gtcgactacgtaactagt ggtggaggc LFSQA coding sequences; HEV ORF2 C-terminal genes are inserted into multiple cloning sites of the HAV 2A-2B joining region, HAV susceptible cells are transfected after in vitro transcription of recombinant RNA (ribonucleic acid), and the recombinant HAV and the like carrying HEV ORF2 antigen genes are rescued. The recombinant virus identification conforms to preparation and verification regulation of a live attenuated hepatitis a vaccine.
Description
Technical field
The invention belongs to the method with preparation vaccine.
Technical background
In public health, hepatitis A and hepatitis E are a kind of main communicable diseases, and this disease has the identical approach through fecal-oral transmission, are subject to kind of a crowd using grownup especially the elderly with pregnant woman as similar Susceptible population and vaccine.The hepatitis A virus (HAV) (HAV) corresponding with hepatitis A and hepatitis E and Hepatitis E virus (HEV) are all picornaviruss, and at present, two kinds of diseases have univalent vaccine.Wherein, hepatitis A vaccine is viral inactivation vaccine and attenuated live vaccine mainly, and viral hepatitis type E vaccine is recombinant vaccine, and antigenic component is that the Hepatitis E virus ORF2 C of prokaryotic expression holds epitope.
The P2 structural domain 454-end being positioned at ORF2 C end during Guu TS etc. adopt with antigen is most important viral neutralizing site, and on P2 structural domain, have 3 orientations outwardly, the ring AA482 – 490 of high conservative, 550 – 566, and 583 – 593, namely with virus-antibody in conjunction with relevant (Guu TS, Liu Z, Ye Q, et al. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. Proc Natl Acad Sci U S A. 2009. 106 (31): 12992-7.).The P2 structural domain of this gene engineering expression can be formed has good immunogenic viruslike particle (Virus Like Particle, VLP).Combined vaccine is the developing direction of vaccine research, provides the significance of both first viral hepatitis type E inflammation combined vaccine to be: combined vaccine has simplification immune programme for children, alleviates by kind person's spirit and economical load, reduces and leaks the rate of kind, be beneficial to the Prevention and controls of two kinds of diseases.Traditional combined vaccine developing thought is that two kinds of existing vaccines are carried out coupling, sharply increases vaccine cost, also cannot solve the compatibility problems between two kinds of vaccines.
The research of the people such as Gerardo shows, HAV genomic 2A-2B joining region and 5 ' non-coding region passable
Hold exogenous genetic fragment (the Konduru K within 600nt, Nakamura SM, Kaplan GG. Hepatitis A virus (HAV) packaging size limit. Virol J. 2009. 6:204.), and can with virus replication, express foreign protein, this is found to be exploitation is that the recombinant combined vaccine of carrier is laid a good foundation with hepatitis A virus (HAV).
The result of study of the people such as Dong Chen shows that hav inactivated vaccine can strengthen the immunogen of viral hepatitis type E recombinant vaccine
Property (Dong C, Dai X, Meng JH. The first experimental study on a candidate combined vaccine against hepatitis A and hepatitis E. Vaccine. 2007. 25 (9): 1662-8.).Our early-stage Study finds that first penta inosculating antibody proper energy forms viruslike particle (VLP), effectively induction of neutralizing antibody (the Kui X of anti-HAV and HEV, Sun M, Xie T, et al. The expression, purification, and immunogenicity of a new chimeric virus-like particle. Viral Immunol. 2009. 22 (1): 49-56.), this result of study has established solid foundation for developing first penta recombiant vaccine.
Summary of the invention
The present invention aims to provide the preparation method of the scorching combined vaccine of a kind of genetically engineered restructuring first viral hepatitis type E, the method is to carry the basis of restructuring hepatitis A virus (HAV) as the scorching combined vaccine of first viral hepatitis type E of viral hepatitis type E antigen epitope genes, viral hepatitis type E antigen is made to be present in hepatitis A virus gene group with the form of gene, and with hepatitis A attenuated strain copying and express, compatibility two kinds of different vaccines preferably in vivo.
Meanwhile, the present invention does not change the scorching combined vaccine of hepatitis A vaccine production method and production first viral hepatitis type E, and production cost is low.
Above-mentioned purpose of the present invention is realized by following preparation method, comprises the following steps:
(1) HAV reverse transcription is gone out full-length cDNA be inserted in carry T7 promotor transcription vector on, and
The HRV 3CP of its 2A-2B joining region is cut and is built 3 glycine peptide chain ggtggcgga, multiple clone site gtcgactacgtaactagt and extra HRV 3CP in order after site and cut site amino acids peptide chain LFSQA encoding sequence, this LFSQA encoding sequence can be the arbitrary combination that HRV 3CP cuts the corresponding all synonym in site, and the auxiliary sequencel arrangement of insertion is as follows:
Ggtggcgga gtcgactacgtaactagt ggtggaggc LFSQA encoding sequence;
(2) HEV cAg ORF2 gene is inserted the multiple clone site place constructed by the genomic 2A-2B joining region of HAV, obtain the HAV full-length genome transcription vector being fitted together to HEV ORF2 antigen epitope genes;
(3) in-vitro transcription, produces restructuring HAV RNA:
A, use restriction endonuclease are at the HAV ic downstream linearized plasmid templates of transcription vector;
B, use in-vitro transcription test kit are transcribed linearizing template, produce restructuring HAV RNA;
(4) the restructuring HAV RNA of phenol chloroform purifying in-vitro transcription;
(5) by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, rescue restructuring HAV virus, increases;
(6) restructuring HAV viruses indentification.
Described method is further: the multiple clone site gtcgactacgtaactagt described in step (1) is the sequence in the restriction endonuclease site do not contained in transcription vector and HEV cAg encoding sequence.
Described method is further: the most preferably encoding sequence that the extra HRV 3CP described in step (1) cuts site amino acids peptide chain LFSQA is that HAV genome has sequence by oneself, and this sequence is ctgttttcacaagcc.
Described method is further: the preferred sequence of step (2) HEV cAg is ORF2 aa 431-615.
Described method is further: step (5) saves restructuring HAV virus by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, comprising:
The HAV permissive cell cell DMEM converging rate without dual anti-culture medium culturing to 90% washes 2 times; Add Opti-MEM 37 DEG C and hatch 30 minutes; It is solution I that the RNA 8ug of purifying adds Opti-MEM to 250ul; It is solution II that Lip2000 adds Opti-MEM to 250ul; Solution I is instilled solution II and is mixed into solution III, incubated at room 5min; Mixed liquor I II is added dropwise to the HAV permissive cell cell containing Opti-MEM, hatches 5-6 hour for 37 DEG C; Replace medium to without dual anti-growth media; 3 days change growth media is afterwards maintenance medium, within every 5 days, changes liquid 1 time; Freeze thawing ultrasonic results restructuring HAV virus after 4 weeks.
Described method is further: its amplification of step (5), after the restructuring HAV virus of rescue gained and HAV virus permissive cell 37 DEG C adsorbs 2h, adds maintenance medium, then is placed in 35 DEG C and cultivates and within 28 days, receive malicious, and period every 5-7 days changes a maintenance medium.
The one that described method is any is: be Huh7 or vero or KMB17 or COS-7 or FRhK-4 by the HAV permissive cell of liposome-mediated restructuring HAV RNA transfection.
The positively effect that the present invention has is:
After the live attenuated virus of the first viral hepatitis type E inflammation associating of the present invention's restructuring enters human body, along with restructuring HAV copying in vivo, HEV opening code-reading frame 2-HEV ORF2 aa 431-615 gene also will be expressed.This is because foreign gene two ends are that HRV 3CP cuts site, while HRV 3CP carries out shearing to HAV structural polypeptide, is also discharged from 2A-2B joining region by HEV exogenous peptide, and this release makes the assembling of hepatitis A virus (HAV) by the impact of exogenous peptide.Meanwhile, the HEV antigen Toplink discharged correctly folds in eukaryotic cell, forms natural conformational epitope peptide.Because HEV exogenous peptide is present in restructuring HAV viral genome with the form of gene, with restructuring HAV attenuated strain copying and express in vivo, avoid the compatibility problems of two kinds of different vaccines.The recombinant virus that the present invention saves out carries out restructuring HAV viruses indentification result as Fig. 2,3, and its virus titer detects the cell culture method (CCID met in " Products in China code " (version in 2000) Attenuated Hepatitis A Vaccine,Live manufacture and vertification regulation
50) measure requirement.
The present invention gets final product the scorching combined vaccine of production first viral hepatitis type E when not changing hepatitis A vaccine production method, more traditional combined vaccine, significantly can reduce production cost.
Accompanying drawing explanation
Fig. 1 is the HAV in-vitro transcription product carrying viral hepatitis type E antigen gene.A is RNA molecule amount marker, B is restructuring HAV RNA.
Fig. 2 is the specific band of the special RT-PCR qualification in restructuring HAV 2A-2B district.A is DNA molecular amount marker, B is the restructuring HAV viral genome 2A-2B district specific band inserting HEV ORF2 antigen gene, and C is the HAV virus 2A-2B district specific band not inserting HEV ORF2 antigen gene.
Fig. 3 ~ 8 are HAV specific immunofluorescence.Wherein: Fig. 3,4 and 5 is the different imagings under the Huh7 cell the same visual field of restructuring HAV virus infection.Fig. 6,7 and 8 is the different imagings of Huh7 cell controls under the same visual field not infecting HAV virus.
Fig. 3 is the anti-HAV immunofluorescence of specificity, and primary antibodie uses the anti-HAV specific antibody of rabbit, and the anti-rabbit IgG of two anti-use red fluorescence marks, visible cells infected endochylema has specificity fluorescent to produce.
Fig. 4 is that 4', 6-diamidino-2-phenylindone (DAPI) is redyed.
Fig. 5 is the superposition composite diagram of Fig. 3 and 4.
Fig. 6 is the contrast of specificity anti-HAV immunofluorescence, and primary antibodie and two anti-ly uses consistent with Fig. 3, and visible compared with control cells endochylema produces without specificity fluorescent.
Fig. 7 is that 4', 6-diamidino-2-phenylindone (DAPI) is redyed.
Fig. 8 is the superposition composite diagram of Fig. 6 and 7.
Fig. 9 ~ 14 are HEV specific immunofluorescence.Wherein, Fig. 9,10 and 11 is the different imagings under the Huh7 cell the same visual field of restructuring HAV virus infection; Figure 12,13 and 14 is the different imagings of Huh7 cell controls under the same visual field not infecting HAV virus.
Fig. 9 is specificity anti-HEV ORF2 immunofluorescence, and primary antibodie uses rabbit anti-HEV ORF2 specific antibody, and the anti-rabbit IgG of two anti-use red fluorescence marks, visible cells infected endochylema has specificity fluorescent to produce.
Figure 10 is that DAPI redyes.
Figure 11 is the superposition composite diagram of Fig. 9 and 10.
Figure 12 is the contrast of specificity anti-HEV ORF2 immunofluorescence, and primary antibodie and two anti-uses consistent with Fig. 9, and visible compared with control cells endochylema produces without specificity fluorescent.
Figure 13 is that DAPI redyes.
Figure 14 is the superposition composite diagram of Figure 12 and 13.
The HAV full-length genome transcription vector that be fitted together to HEV ORF2 antigen epitope genes of Figure 15 for HEV cAg ORF2 C end group is obtained because of the multiple clone site place inserted constructed by the genomic 2A-2B joining region of HAV.
Below by way of embodiment, technical scheme of the present invention is described further.Embodiment comprises but does not limit the scope of the invention.
Embodiment
(1) preparation method of the scorching combined vaccine of genetically engineered restructuring first viral hepatitis type E
Embodiment 1:
(1) HAV reverse transcription is gone out full-length cDNA be inserted in carry T7 promotor transcription vector on, and
The HRV 3CP of its 2A-2B joining region is cut and is built 3 glycine peptide chain ggtggcgga, multiple clone site gtcgactacgtaactagt and extra HRV 3CP in order after site and cut site amino acids peptide chain LFSQA encoding sequence, this LFSQA encoding sequence can be the arbitrary combination that HRV 3CP cuts the corresponding all synonym in site, and the auxiliary sequencel arrangement of insertion is as follows:
Ggtggcgga gtcgactacgtaactagt ggtggaggc LFSQA encoding sequence;
(2) HEV cAg ORF2 gene is inserted the multiple clone site place constructed by the genomic 2A-2B joining region of HAV, obtains the HAV full-length genome transcription vector being fitted together to HEV ORF2 antigen epitope genes:
(3) in-vitro transcription, produces restructuring HAV RNA:
A, use restriction endonuclease are at the HAV ic downstream linearized plasmid templates of transcription vector;
B, use in-vitro transcription test kit are transcribed linearizing template, produce restructuring HAV RNA;
(4) the restructuring HAV RNA of phenol chloroform purifying in-vitro transcription;
(5) by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, rescue restructuring HAV virus, increases;
(6) restructuring HAV viruses indentification.
Embodiment 2:
In embodiment 1 method, except the following technical scheme of step (1), all the other steps are identical with embodiment 1.This technical scheme is:
Multiple clone site gtcgactacgtaactagt described in step (1) is the sequence in the restriction endonuclease site do not contained in transcription vector and HEV cAg encoding sequence, as long as the restricted accounting restriction enzyme site do not contained in transcription vector and HEV cAg encoding sequence all can be used as the multiple clone site of insertion.
Embodiment 3:
In embodiment 1 method, the most preferably encoding sequence cutting site amino acids peptide chain LFSQA except the extra HRV 3CP described in step (1) is that HAV genome has sequence by oneself, and this sequence is outside ctgttttcacaagcc, and all the other steps are identical with embodiment 1.
Embodiment 4:
In embodiment 1 method, the preferred sequence except step (2) HEV cAg is that except ORF2 aa 431-615, all the other steps are identical with embodiment 1.
Embodiment 5:
In embodiment 1 method, except step (5) is saved except restructuring HAV virus by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, all the other steps are identical with embodiment 1.This step (5) comprising:
The HAV permissive cell cell DMEM converging rate without dual anti-culture medium culturing to 90% washes 2 times; Add Opti-MEM 37 DEG C and hatch 30 minutes; It is solution I that the RNA 8ug of purifying adds Opti-MEM to 250ul; It is solution II that Lip2000 adds Opti-MEM to 250ul; Solution I is instilled solution II and is mixed into solution III, incubated at room 5min; Mixed liquor I II is added dropwise to the HAV permissive cell cell containing Opti-MEM, hatches 5-6 hour for 37 DEG C; Replace medium to without dual anti-growth media; 3 days change growth media is afterwards maintenance medium, within every 5 days, changes liquid 1 time; Freeze thawing ultrasonic results restructuring HAV virus after 4 weeks.
Embodiment 6:
In embodiment 1 method, except step (5), it increases is, after the restructuring HAV virus of saving gained is adsorbed 2h with HAV virus permissive cell 37 DEG C, add maintenance medium, then be placed in 35 DEG C of cultivations, 28 days receipts poison, period every 5-7 days changes outside a maintenance medium, and all the other steps are identical with embodiment 1.
In method described in above embodiment 1 ~ 6, be any one among Huh7 or vero or KMB17 or COS-7 or FRhK-4 by the HAV permissive cell of liposome-mediated restructuring HAV RNA transfection.
(2) restructuring HAV titer determination
According to the cell culture method (CCID in " Products in China code " (version in 2000) Attenuated Hepatitis A Vaccine,Live manufacture and vertification regulation
50) carry out virus titer mensuration, specifically:
1. get and obtain restructuring HAV virus and do 10 times of serial dilutions;
2. every extent of dilution inoculates 4 bottles of (25cm
2) grow up to vero or KMB17 or other HAV permissive cells of individual layer, every bottle graft kind 1ml viral dilution liquid, add maintenance medium to 8ml after 37 DEG C of absorption 2h;
3. cultivate 25-26 days for 35 DEG C, period every 5-7 days changes a maintenance medium;
4. arrive after date every bottle 0.4ml 0.01mol/L pH7.4 PBS solution and receive poison, after freeze thawing is ultrasonic, high speed centrifugation removes cell debris, obtains virus liquid;
5. obtained virus liquid ELISA method is detected HAV virus antigen;
6. with Reed-Muench formulae discovery CCID
50, virus titer should be not less than 6.50logCCID
50.
(3) qualification restructuring HAV virus
(1) adopt viral RNA to extract test kit, at HAV 2A-2B district design Auele Specific Primer, this Auele Specific Primer is HAV 2A-2B joining region upstream and downstream arbitrary sequence, carries out RT-PCR, and agarose gel electrophoresis observes visible specific band (as Fig. 2).
(2) adopt HAV specific antibody and HEV ORF2 specific antibody, carry out Immunofluorescence test, after result display restructuring HAV virus infection, create the special immunofluorescence of HAV and HEV (as Fig. 9-Figure 14).
Claims (7)
1. a preparation method for the scorching combined vaccine of genetically engineered restructuring first viral hepatitis type E, comprises the following steps:
(1) HAV reverse transcription is gone out full-length cDNA be inserted in carry T7 promotor transcription vector on, and
The HRV 3CP of its 2A-2B joining region is cut and is built 3 glycine peptide chain ggtggcgga, multiple clone site gtcgactacgtaactagt and extra HRV 3CP in order after site and cut site amino acids peptide chain LFSQA encoding sequence, this LFSQA encoding sequence can be the arbitrary combination that HRV 3CP cuts the corresponding all synonym in site, and the auxiliary sequencel arrangement of insertion is as follows:
Ggtggcgga gtcgactacgtaactagt ggtggaggc LFSQA encoding sequence;
(2) by HEV cAg ORF2 C end group because inserting the multiple clone site place constructed by the genomic 2A-2B joining region of HAV, obtain the HAV full-length genome transcription vector being fitted together to HEV ORF2 antigen epitope genes;
(3) in-vitro transcription, produces restructuring HAV RNA:
A, use restriction endonuclease are at the HAV ic downstream linearized plasmid templates of transcription vector;
B, use in-vitro transcription test kit are transcribed linearizing template, produce restructuring HAV RNA;
(4) the restructuring HAV RNA of phenol chloroform purifying in-vitro transcription;
(5) by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, rescue restructuring HAV virus, increases;
(6) restructuring HAV viruses indentification.
2. method according to claim 1, is characterized in that: the multiple clone site gtcgactacgtaactagt described in step (1) is the sequence in the restriction endonuclease site do not contained in transcription vector and HEV cAg encoding sequence.
3. method according to claim 1, is characterized in that: the most preferably encoding sequence that the extra HRV 3CP described in step (1) cuts site amino acids peptide chain LFSQA is that HAV genome has sequence by oneself, and this sequence is ctgttttcacaagcc.
4. method according to claim 1, is characterized in that: the preferred sequence of step (2) HEV cAg is ORF2 aa 431-615.
5. method according to claim 1, is characterized in that: step (5) saves restructuring HAV virus by liposome-mediated restructuring HAV RNA transfection HAV permissive cell, comprising:
The HAV permissive cell DMEM converging rate without dual anti-culture medium culturing to 90% washes 2 times; Add Opti-MEM 37 DEG C and hatch 30 minutes; It is solution I that the RNA 8ug of purifying adds Opti-MEM to 250ul; It is solution II that Lip2000 adds Opti-MEM to 250ul; Solution I is instilled solution II and is mixed into solution III, incubated at room 5min; Mixed liquor I II is added dropwise to the HAV permissive cell cell containing Opti-MEM, hatches 5-6 hour for 37 DEG C; Replace medium to without dual anti-growth media; 3 days change growth media is afterwards maintenance medium, within every 5 days, changes liquid 1 time; Freeze thawing ultrasonic results restructuring HAV virus after 4 weeks.
6. method according to claim 1, it is characterized in that: its amplification of step (5) is after the restructuring HAV virus of rescue gained and HAV virus permissive cell 37 DEG C are adsorbed 2h, add maintenance medium, then be placed in 35 DEG C of cultivations, 28 days receipts poison, period every 5-7 days changes a maintenance medium.
7. the one that method according to claims 1 to 6 is any, is characterized in that: be Huh7 or vero or KMB17 or COS-7 or FRhK-4 by the HAV permissive cell of liposome-mediated restructuring HAV RNA transfection.
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Cited By (2)
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| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
| CN112724205A (en) * | 2021-02-01 | 2021-04-30 | 山西省中医药研究院（山西省中医院） | Method for preparing virus-like particles from hepatitis E virus (HCV) 239 protein and application of virus-like particles |
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| WO2000014263A2 (en) * | 1998-09-03 | 2000-03-16 | Board Of Regents, The University Of Texas System | Recombinant hepatitis a virus (hav), hav variants, hav-based vaccines and methods of producing them |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
| CN109943536B (en) * | 2019-03-26 | 2021-09-14 | 昆明理工大学 | Method for culturing hepatitis E virus and method for preparing inactivated vaccine thereof |
| CN112724205A (en) * | 2021-02-01 | 2021-04-30 | 山西省中医药研究院（山西省中医院） | Method for preparing virus-like particles from hepatitis E virus (HCV) 239 protein and application of virus-like particles |
| CN112724205B (en) * | 2021-02-01 | 2023-05-02 | 山西省中医药研究院（山西省中医院） | Method for preparing virus-like particles from C hepatitis E virus 239 protein and application thereof |
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