CN101497891A - Novel recombinant viral vector and use thereof - Google Patents
Novel recombinant viral vector and use thereof Download PDFInfo
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- CN101497891A CN101497891A CNA2008100653591A CN200810065359A CN101497891A CN 101497891 A CN101497891 A CN 101497891A CN A2008100653591 A CNA2008100653591 A CN A2008100653591A CN 200810065359 A CN200810065359 A CN 200810065359A CN 101497891 A CN101497891 A CN 101497891A
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Abstract
The invention relates to a gene engineering technology, in particular to a novel recombination viral vector comprising a viral vector and a nucleotide sequence; wherein the nucleotide sequence is used for coding the fused protein of two antigens of a pathogenic virus, and each antigen comprises a B cell epitope and a T cell epitope. The recombination vector enables the immune animals to produce a humoral immunity reaction and a cellular immunity reaction, thereby achieving the aim of preventing and treating diseases. The injection of the recombination adenoviruses carrier has high safety.
Description
Technical field
The present invention relates to genetic engineering technique, especially relate to a kind of recombinant chou with coding antigenic nucleotide sequence of Causative virus and virus vector sequence construct.
Background technology
Hepatitis B is that (Human Hepatitis B virus HBV) causes, HBV is a dna double chain virus, mainly through blood, contact and mother and baby's vertical transmission closely by the viruses of human hepatitis B.Different areas, infection probability and the circulation way of HBV also are not quite similar.After infecting HBV, part patient will be developed into chronic persistent infection state, and what have may develop into liver cirrhosis or primary hepatocellular carcinoma.At present, according to estimates, about 3.5 hundred million people of whole world hepatitis B carrier, area, Asia about 2.2 hundred million.It is hepatitis B virus carrierss that China has 1.3 hundred million people approximately, hundred million yuan of about 300-500 of medical expense that are used for the treatment of hepatitis every year, and most infect occur in the newborn infant or the Childhood, because how asymptomatic the infection that this stage takes place is in early days, acute phase extremely difficulty be found, therefore will develop into the chronic viral hepatitis B patient more than 90%.At present, for chronic viral hepatitis B patient and hepatitis B antigen surface carrier, still there is not effective methods of treatment at present.Though limited chemotherapy can suppress hbv replication to a certain extent, can not thoroughly remove intravital infective virus.Discover that the different final results of the acute and chronic hepatopathy of hepatitis B patient are because due to the immune response difference that body infects HBV to a great extent.In acute hepatitis b patient body, at the cytotoxic T lymphocyte of HBV (cytotoxicity T lymphocyte, CTL) reaction be very active, be multidigit point and high degree of specificity, can remove infective virus rapidly; And reply obviously low in chronic viral hepatitis B patient and the hepatitis B virus healthy carrier body at the specific CTL of HBV, cause virus to continue to exist, therefore academia thinks that correcting and overcome the intravital CTL low reaction of patient state may be that body is removed HBV, the key that the patient is thoroughly recovered at present.
Does CTL reach the purpose of removing HBV by what mechanism? because HBV cccDNA structure is special, has secular stability again, think that CTL has only the liver cell by destroying and kill and wound infection just can reach the effect of removing virus at first always.But discovering in recent years is true really not so.In HBV transgenic mouse model, the CTL of virus-specific mainly removes virus by the pathogenic process of acellular, the removing of having only the remaining virus of a minimum part is to realize impaired liver cell less than 1% in the whole virus sweep process by the apoptosis and the cell-cytotoxic reaction of CTL mediation.Guidotti finds also that in HBV acute infection chimpanzee animal model in acute phase, the HBV DNA overwhelming majority is eliminated, and hepatic injury, apoptosis and ALT do not raise during this.This shows that in HBV acute infection process HBV mainly removes by the pathogenic mechanism of this acellular.Its detailed process is: the viral target antigen on the specific CTL identification of HBV infected liver cell surface, discharge cytokine immediately, mainly be IFN-γ and TNF-α, directly act on the HBV infected liver cell, activate the antiviral molecule mechanism in the liver cell by paracrine mechanism.Simultaneously, these cytokines activate the Kupffer cell, and further produce more TNF-α by positive feedback mechanism.The environment of this cytokine causes the degraded of intracellular virus particle and viral RNA.
The Hepatitis B virus vaccine of using on the market is the yeast reconstituted hepatitis B vaccine at present, is mainly used in the prevention of hepatitis B, can not play the effect of treatment hepatitis B.Cai Xiongmao etc. 2002, express HBV core and preceding S1 fusion rotein intestinal bacteria and eucaryon.Chen Xinchun etc. 2003, with hepatitis B core antigen and pre S 1 antigen fusion rotein inducing mouse, make it produce immune response.
The therapeutic hepatitis B vaccine that enters the clinical I phase abroad and be in laboratory stage both at home and abroad all belongs to dna vaccination, the problem that dna vaccination exists at present is whether exogenous nucleic acid can be incorporated into and cause canceration in the karyomit(e), can cause that problems such as immunopathogenesis effect do not have definite conclusion as yet, and the vaccine immune response intensity of bringing out a little less than.So the adenovirus of answering wide, the efficient transduction of apparatus host range, advantage such as stable, safe, easy to operate then can be avoided above-mentioned shortcoming as carrier.
Adenovirus is extensive in distributed in nature, all finds its existence in many Mammalss and domestic animals.So far the various adenovirus of different serotypes more than 100 kinds have been separated to.Adenovirus is the icosahedron viruses shell structures, and its genome is linear double-stranded DNA.Because adenovirus is as the advantage that carrier has that high efficiency of infection and high exogenous gene expression level, the preparation of high titre recombinant virus simultaneously are simple, capacity is fit to load most of foreign genes, unconformability is gone into target cell genome or the like, so adenovirus is widely used in Vectors in Gene Therapy.
Summary of the invention
For addressing the above problem, the object of the present invention is to provide a kind of novel recombinant viral vector.
For achieving the above object, technical scheme of the present invention is: a kind of novel recombinant viral vector, this carrier comprises virus vector and a nucleotide sequence, two antigenic fusion roteins of described nucleotide sequence coded Causative virus, described antigen contains B cell epitope and t cell epitope, and this recombinant vectors can make immune animal produce humoral immunization and cell immune response.
Described virus vector is adenovirus carrier, gland relevant viral vector, SV40 virus vector, retrovirus vector, herpes simplex virus vector or vaccinia virus vector.
Described adenovirus carrier is a disappearance sexual gland virus carrier.
Described nucleotide sequence is inserted into E1, E2, E3 or the E4 zone of adenovirus.
Described adenovirus carrier is the adenovirus hominis carrier.
Described adenovirus carrier is people's 5 type adenovirus carriers, 35 type adenovirus carriers, 2 type adenovirus carriers or 6 type adenovirus carriers.
Described adenovirus carrier is the animal adenovirus carrier.
Described Causative virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus or hepatitis E virus.
Described nucleotide sequence is the nucleotide sequence of coding hepatitis B virus antigen epi-position.
The antigen of described Causative virus is surface antigen, cAg, pol antigen of hepatitis B virus or the antigen that contains t cell epitope, in the described nucleotide sequence coded above-mentioned antigen two kinds.
The fusion rotein of described nucleotide sequence coded hepatitis B virus surface antigen and cAg, two antigenic nucleotide sequences can connect by one section nucleotide sequence, also can directly connect.
The fusion rotein of described nucleotide sequence coded hepatitis B virus surface antigen and cAg is shown in SEQ IDNO:1.
Described Causative virus is human papillomavirus, avian influenza virus, sars virus or HIV virus.
Described recombinant vectors also contains at least one immunologic stimulant gene order.
Described immunologic stimulant is interleukin-, Interferon, rabbit, granulocyte colony-stimulating factor or granulocyte-macrophage colony stimutaing factor.
Above-mentioned novel recombinant viral vector is used for preventing and treating the application of the medicine of the disease that is caused by Causative virus in preparation.
The vaccine of the above-mentioned novel recombinant viral vector preparation of a kind of usefulness.
Novel recombinant adenovirus of the present invention obtains by the following method, be that two or more goal gene are merged by gene engineering method, be inserted in the adenovirus carrier through improveing with enzyme butt formula then and form recombinant chou, in intestinal bacteria, increase then, behind separation, extraction, purifying, rotaring redyeing 293 cell (HEKC) is at the viruslike particle of 293 cell internal packings one-tenth.
According to such scheme, at first obtain required goal gene by pcr amplification, two goal gene HBc are connected by the pcr amplification method with HBs, obtain having the new fragment of HBc and HBs gene.This target gene fragment that newly obtains is cloned into shuttle vector pDC515 (io), in 293 cells, obtains recombinant adenovirus by the FLP-frt recombinase system.
Hepatitis B virus core antigen and hepatitis B surface antigen have the effect that causes ctl response and humoral immunoresponse(HI).Act on animal capable with hepatitis B virus core antigen and surface antigen as the recombinant chou of goal gene and cause ctl response and humoral immunoresponse(HI) in the animal body.The described recombinant adenoviral vector of the application acts on the combination that the ctl response intensity that causes behind the animal is far longer than hepatitis B virus core antigen recombinant adenoviral vector and surface antigen recombinant adenoviral vector, also be better than the recombinant chou of independent hepatitis B virus core antigen, because adenovirus has the advantage that high efficiency of infection and high exogenous gene expression level, unconformability are gone into target cell genome or the like as carrier, therefore injecting recombinant adenoviral vector of the present invention has tight security.In addition, also can cause very strong ctl response with hepatitis B virus core antigen, surface antigen and other antigen as the recombinant adenoviral vector of goal gene.Therefore, inject recombinant viral vector of the present invention after, can make to cause humoral immune reaction and cell immune response in the animal body, thus reach the prevention and the treatment disease purpose.
Description of drawings
Fig. 1 is that the CS9 recombinant adenovirus vaccine makes up schema.
Fig. 2 is pDC515 (io) plasmid map.
Fig. 3 is a PBHG Δ E1.3flp plasmid map.
Fig. 4 is the electrophoresis picture that PCR method detects recombinant adenovirus HB-CS.
Fig. 5 respectively organizes 1,4 all ctl response intensity tables (n=3) behind the injected in mice recombinant adenovirus.
Embodiment
The structure of embodiment 1, CS9 recombinant adenovirus vaccine
Below in conjunction with Fig. 1 the structure of CS9 recombinant adenovirus vaccine is done and to be described in further detail.
The following stated 5 type adenovirus hominiss can also be 2 type adenovirus hominiss, 6 type adenovirus hominiss, 35 type adenovirus hominiss or animal adenovirus.
1, the acquisition of the nucleotide sequence HBs of coding hepatitis B virus surface antigen: extracting the hepatitis B patients serum, extract hepatitis B virus DNA from the patients serum, is template with the DNA that extracts, the design primer:
HBV-S-F：5′-ATCTCAATGT?GAGAGCACAAC-3′，
HBV-S-R:5 '-TCAAATGTATACCCAAAGAC-3 ' (China's big gene Shanghai ancient cooking vessel peace is synthetic), carry out pcr amplification, amplification condition is: 94 ℃ of sex change of first circulation 2 minutes, each circulation later on: 94 ℃ of sex change 30 seconds, 49 ℃ of annealing 30 seconds, 72 ℃ were extended 1 fen, totally 35 circulations, 72 ℃ were extended 7 minutes afterwards, obtained the HBs fragment and got, and its concrete sequence is shown in SEQ ID NO:2.
2, the acquisition of the nucleotide sequence HBc of coding hepatitis B core antigen: extracting the hepatitis B patients serum, extract hepatitis B virus DNA from the patients serum, is masterplate with the DNA that extracts, the design primer:
HBV-C-F：5′-GCTAGCATGGA?CATTGACCCGTAT-3′，
HBV-C-R:5 '-GTTGTGCTCTCACATTGAGAT-3 ' (China's big gene Shanghai ancient cooking vessel peace is synthetic), carry out pcr amplification, amplification condition is: 94 ℃ of sex change of first circulation 2 minutes, each circulation later on: 94 ℃ of sex change 30 seconds, 49 ℃ of annealing 30 seconds, 72 ℃ were extended 1 fen, totally 35 circulations, 72 ℃ were extended 7 minutes afterwards, obtained the HBc fragment, and its concrete sequence is shown in SEQ ID NO:3.
3, the segmental acquisition of CS fusion gene: will go up HBc fragment and the mixing of HBs fragment that two steps obtained earlier, add dNTPs again, the rTaq enzyme, damping fluid and distilled water, 94 ℃ of sex change 2 minutes, 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 2 fens, after the circulation, add primer HBV-C-F and HBV-S-R again in the PCR system, carry out PCR: 94 ℃ of sex change of first circulation 2 minutes, each circulation later on: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 49 ℃, 72 ℃ were extended 1.5 fens, totally 35 circulations, and 72 ℃ were extended 7 minutes afterwards.Obtain CS fusion gene fragment, its concrete sequence is shown in SEQ ID NO:1.Its PCR fragment that obtains is inserted in the T carrier (Promega company), obtains the T/CS carrier.
4, the acquisition of adenovirus shuttle vector PDC515/CS: obtain the CS fragment with NheI and SalI digested plasmid T/CS; Cut carrier PDC515 (io) (Microbix Biosystems company) with NheI and SalI enzyme, its plasmid map as shown in Figure 2, obtain the PDC515/NheI+SalI fragment, connect above-mentioned two fragments with the T4DNA ligase enzyme, obtain carrying the shuttle plasmid pDC515/CS of hbsag gene HBs and cAg gene HBc, called after adenovirus shuttle vector PDC515/CS.
5, homologous recombination: adenovirus shuttle vector PDC515/CS and 5 type adenovirus hominis skeleton carrier PBHG Δ E1.3flp ((Microbix Biosystems companies, its plasmid map as shown in Figure 3, utilize Lipofectamine2000 (invitrogen) cotransfection to 293 cell, by the method acquisition virus of homologous recombination.Virus plaque appearred in cotransfection 11-15 days, through 3 virus plaque purifying, and the recombinant adenovirus that obtains expressing hepatitis B virus core antigen and hepatitis B surface antigen, called after HB-CS recombinant adenovirus vaccine (abbreviating CS9 as).Extract recombinant dna, PCR method detects the CS sequence of inserting, and goal gene detects primer and is: primer HBV-C-F and HBV-S-R, adenovirus carrier detect primer and are
E2BF：5’-TCG?TTT?CTC?AGC?AGC?TGT?TG?3′，
E2BR：5’-CAT?CTG?AAC?TCA?AAG?CGT?GG?3′；
PDC515F：5′ACA?TCC?ACT?TTG?CCT?TTC?TC?3′，
PDC515R:5 ' GAC, AAA, CCA, CAA, CTA, GAA, TGC3 ' (China's big gene Shanghai ancient cooking vessel peace), recombinant adenovirus is identified qualification result is seen Fig. 4: swimming lane 1 is DL2000marker, and swimming lane 2-4 is a primer with E2BF and E2BR: 2 positive contrasts, 3 is sample, 4 negative contrasts, swimming lane 5-7 is a primer with pDC515F and pDC515R: 5 positive contrasts, 6 is sample, 7 negative contrasts, swimming lane 10-12 is a primer with HBV-C-F and HBV-S-R: 10 is sample, 11 positive contrasts, 12 negative contrasts.
Check order (China's big gene Shanghai ancient cooking vessel peace) the adenovirus called after adenovirus vaccine HB-CS (abbreviating CS9 as) that sequencing result is correct simultaneously.
The recombinant adenovirus amplification
Cultivate 293 cells (HEKC), the inoculation recombinant adenovirus increases in 293 cells.Amplification method is known in the art, can be with reference to " tissue culture and molecular cell learn a skill " (E Zheng Beijing Publishing House).
The recombinant adenovirus purifying
Adopt ion exchange column HPLC purification system that recombinant adenovirus is carried out purifying.
The recombinant adenovirus determination of activity
The ratio titre that the TCID50 method records recombinant adenovirus CS9 is 4.80%.
The adenovirus vaccine CS9 that has integrated hepatitis B virus core antigen gene and hbsag gene can simulate normal adenovirus infection pattern behind infection animal, the induction of immunity system produces specific cellular immunity and the humoral immunization at hepatitis B virus core antigen, when body contacts pathogenic agent once more, stimulate specific immunity memory cell secrete cytokines γ-IFN by many signal pathways, γ-IFN has been verified to have antiviral effect.Thereby the ELISPOT experiment is promptly by detecting the secretion indirect detection specific CTL reaction of γ-IFN.Recombinant adenovirus CS9 immunity C57/BL mouse, (construction process of C7 and S8 is with reference to the construction process of asking described recombinant adenovirus CS9 in the basis to use the recombinant adenovirus S8 that contains the recombinant adenovirus C7 of HBc gene separately and contain the HBs gene separately simultaneously, be not described in detail herein) make up (C7/S8) as contrast, get its spleen in the 1st, 4 weeks and prepare single splenocyte suspension, be the stimulated in vitro thing with hepatitis B virus core antigen polypeptide and hepatitis B surface antigen polypeptide respectively, carry out the ELISPOT test.Under the same terms, the mice spleen cell of seeing test group secretion γ-IFN promptly forms the cell of spot, and (whether spot forming cell SFC) increases than control group is remarkable.
Experiment is divided into three groups, injects CS9 respectively, and C7/S8 and virus are preserved liquid, 6 C57/BL mouse of every treated animal injection (male, 6-8 age in week, Zhongshan University experimental animal center).Back leg intramuscular injection 100ul/, CS9 group: 10
10The vp/ mouse, C7/S8 group: C710
10Vp+S810
10The vp/ mouse.3 sacrifice of animal are got for every group during 1,4 weeks in the injection back.
The humoral immunization experiment: pluck eyeball and get whole blood, 5000rpm is the sucking-off upper serum after centrifugal 10 minutes, carries out ELISA experiment (being undertaken by Shanghai Shiye Kehua Biotechnology Co., Ltd's specification sheets) after the 1:2 dilution, surveys the HBV surface antibody.The result shows that 1,4 all experimental mice and the anti-HBV surface antibody of injection C7/S8 group mice serum are all positive, and it is all negative that injecting virus is preserved the liquid group.Induced humoral immune reaction after promptly injecting adenovirus, illustrated that adenovirus vaccine of the present invention has the effect of prevention hepatitis B.
ELISPOT experiment: the aseptic spleen of getting grinds and makes the individual cells suspension behind the sacrifice of animal, dilute, with it be added on bag in advance by and the good ELISPOT plate of sealing in 96 holes, every hole 5 * 10
5Individual splenocyte.Add hepatitis B virus core antigen polypeptide and hepatitis B surface antigen polypeptide (big gene Shanghai Tian Yuan is synthetic in China) as stimulator.Carry out ELISPOT experiment concrete steps with reference to seeing U-Cytech test kit specification sheets.
Test group is carried hepatitis B virus core antigen epi-position and surface antigen epi-position simultaneously, has advantage on the ctl response inducing.By Fig. 5 result as can be known the ctl response intensity of test group be higher than control group far away.
As from the foregoing: after specific polypeptide stimulates, can discharge γ-IFN external behind the injected in mice recombinant adenovirus CS9, thereby proof recombinant adenovirus CS9 can induce body to produce specific cell immunoreaction, when the activated cell runs into identical pathogenic agent once more, will discharge cytokine performance antivirus actions such as γ-IFN.
Recombinant adenovirus CS9 of the present invention thus can produce reaction of HBV cAg specific CTL and the reaction of HBV surface antigen specific CTL by inducing mouse, thereby prompting recombinant adenovirus CS9 has better therapeutic action to hepatitis B.
SEQUENCE?LISTING
＜110〉Shenzhen Yuanxing Bio-Pharm Co., Ltd.
＜120〉a kind of novel recombinant viral vector and application thereof
<160>3
<170>PatentIn?version3.3
<210>1
<211>1227
<212>DNA
＜213〉hepatitis B virus
<400>1
<210>2
<211>688
<212>DNA
＜213〉hepatitis B virus
<400>2
<210>3
<211>566
<212>DNA
＜213〉hepatitis B virus
<400>3
Claims (16)
1. novel recombinant viral vector, it is characterized in that: this carrier comprises virus vector and a nucleotide sequence, two antigenic fusion roteins of described nucleotide sequence coded Causative virus, described antigen contains B cell epitope and t cell epitope, and this recombinant vectors can make immune animal produce humoral immunization and cell immune response.
2. novel recombinant viral vector according to claim 1 is characterized in that: described virus vector is adenovirus carrier, gland relevant viral vector, SV40 virus vector, retrovirus vector, herpes simplex virus vector or vaccinia virus vector.
3. novel recombinant viral vector according to claim 2 is characterized in that: described adenovirus carrier is a disappearance sexual gland virus carrier.
4. novel recombinant viral vector according to claim 3 is characterized in that: described nucleotide sequence is inserted into E1, E2, E3 or the E4 zone of adenovirus.
5. novel recombinant viral vector according to claim 4 is characterized in that: described adenovirus carrier is the adenovirus hominis carrier.
6. novel recombinant viral vector according to claim 5 is characterized in that: described adenovirus carrier is people's 5 type adenovirus carriers, 35 type adenovirus carriers, 2 type adenovirus carriers or 6 type adenovirus carriers.
7. novel recombinant viral vector according to claim 2 is characterized in that: described adenovirus carrier is the animal adenovirus carrier.
8. novel recombinant viral vector according to claim 1 is characterized in that: described Causative virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus or hepatitis E virus.
9. novel recombinant viral vector according to claim 8 is characterized in that: described nucleotide sequence is the nucleotide sequence of coding hepatitis B virus antigen epi-position.
10. novel recombinant viral vector according to claim 9, it is characterized in that: the antigen of described Causative virus is the surface antigen of hepatitis B virus, cAg, pol antigen or contains t cell epitope, in the described nucleotide sequence coded above-mentioned antigen two kinds.
11. novel recombinant viral vector according to claim 10, it is characterized in that: the fusion rotein of described nucleotide sequence coded hepatitis B virus surface antigen and cAg, two antigenic nucleotide sequences can connect by one section nucleotide sequence, also can directly connect.
12. novel recombinant viral vector according to claim 10 is characterized in that: the fusion rotein of described nucleotide sequence coded hepatitis B virus surface antigen and cAg, shown in SEQ ID NO:1.
13. novel recombinant viral vector according to claim 1 is characterized in that: described Causative virus is human papillomavirus, avian influenza virus, sars virus or HIV virus.
14. novel recombinant viral vector according to claim 1 is characterized in that: recombinant vectors also contains at least one immunologic stimulant gene order.
15. novel recombinant viral vector according to claim 14 is characterized in that: described immunologic stimulant is interleukin-, Interferon, rabbit, granulocyte colony-stimulating factor or granulocyte-macrophage colony stimutaing factor.
16. each described novel recombinant viral vector of claim 1-15 is used for preventing and treating the application of the medicine of the disease that is caused by Causative virus in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636359A (en) * | 2016-11-15 | 2017-05-10 | 上海派森诺生物科技股份有限公司 | Sanger-based method for sequencing recombinant HPV (human papillomavirus) adenovirus genome |
| CN106699895A (en) * | 2016-12-05 | 2017-05-24 | 上海科华生物工程股份有限公司 | Novel fusion antigen and detection kit comprising same and application |
-
2008
- 2008-02-03 CN CNA2008100653591A patent/CN101497891A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 周陶友 等: "融合表达HBsAg/HBcAg的重组质粒诱导的小鼠免疫应答", 《世界华人消化杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636359A (en) * | 2016-11-15 | 2017-05-10 | 上海派森诺生物科技股份有限公司 | Sanger-based method for sequencing recombinant HPV (human papillomavirus) adenovirus genome |
| CN106636359B (en) * | 2016-11-15 | 2020-08-18 | 上海派森诺生物科技股份有限公司 | Method for sequencing recombinant HPV adenovirus genome based on sanger sequencing |
| CN106699895A (en) * | 2016-12-05 | 2017-05-24 | 上海科华生物工程股份有限公司 | Novel fusion antigen and detection kit comprising same and application |
| CN106699895B (en) * | 2016-12-05 | 2020-08-04 | 上海科华生物工程股份有限公司 | Novel fusion antigen, detection kit containing same and application |
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Application publication date: 20090805 |