CN102154226B - Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B - Google Patents
Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B Download PDFInfo
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Abstract
The invention relates to the field of a virus, a compound and preparation or purification of the virus, in particular to a chimeric virus of a complete envelope protein of an HCV (hepatitis C virus) and a GB virus B. The chimeric virus is formed by sequentially connecting a 5'-end non-coding region and core protein gene sequence of the GB virus B, a complete envelope protein gene sequence of the HCV and a nonstructural protein and 3'-end non-coding region sequence of the GB virus B, wherein the complete envelope protein gene sequence of the HCV is a complete envelope protein gene sequence of a 1b-genotype HCV. A marmoset monkey can be successfully infected by the virus by carrying out intrahepatic injection and intravenous injection of a primary marmoset monkey serum containing the chimeric virus. The chimeric virus simulates infection and immune states of the HCV in a primate body. A platform for carrying out immunoassay and evaluation on the complete envelope protein of the HCV is provided. The scientific problems of limitation on HCV immunity prevention and treatment research, vaccine evaluation and the like due to shortage of a small primate infection model are solved.
Description
Technical Field
The present invention relates to the field of viruses, compositions and their preparation or purification, particularly viruses modified by the introduction of foreign genetic material.
Background
Hepatitis C Virus (HCV) is one of the main causative agents of acute/chronic Hepatitis transmitted through blood, and the prevalence of HCV infection varies widely around the world, and is most serious in developing countries. Currently, about 1.7 million people worldwide are infected with hepatitis c virus. 50-85% of hepatitis C virus infected patients develop chronic hepatitis, 10-20% of which further develop complex chronic liver diseases such as cirrhosis, and 1-5% of patients develop liver cancer after twenty-three decades. However, there is still a lack of effective therapeutic agents for hepatitis c virus infection, and more seriously, there is no hepatitis c preventive vaccine in the world so far. The research of hepatitis C has not made breakthrough progress until now, and the key factor is that because hepatitis C virus only can infect human and chimpanzee, ideal small primate model infected by hepatitis C virus is lacked, thus greatly limiting the research of immune prevention and treatment of hepatitis C virus.
Hepatitis c virus is a virus of the hepacivirus genus of the flaviviridae family, whose genome consists of structural (core, envelope) and non-structural (protease, helicase, enzyme cofactor) proteins and non-coding regions. The envelope protein (E) dimer (E1E2, containing p7 protein) is directly involved in the interaction with host cells and induces the body to produce neutralizing antibodies and specific cellular immunity, which are main protective antigens. In recent years, cell culture of infectious clones of hepatitis C virus whole genome has succeeded, and the progress of cell culture of hepatitis C virus has been made. However, naturally infected (wild) strains are not yet available for cell culture, and in particular, there is no effective small primate infection model to simulate real hepatitis c virus infection, which still greatly limits the studies on hepatitis c virus immunity.
In the prior art, studies using chimeric viruses have been found in HCV/HIV, HCV/VSV, HCV/YFV, HCV/CSFV, HCV/BVDV and HCV/PV. These constructed HCV chimeric viruses or pseudoviruses (pseudoviruses) are mainly used for studies on interaction between hepatitis c virus proteins and cells, measurement of neutralizing antibodies, gene regulation, and the like. Because these skeletons have no hepatotropic property, they cannot be used in the research of immune prevention and treatment of hepatitis C vaccine.
GB virus B (GBV-B) is a virus that most recently resembles hepatitis C virus, the genus of Hepacivirus of the Flaviviridae family. Unlike hepatitis C virus, GB virus B replicates in small primate marmosets common marmoset (Callithrix jacchuejacacchus) and Pacific monkeys (Tamarin, Saguinus spp) and causes acute or chronic hepatitis. However, the GB virus B still has differences with the hepatitis C virus and cannot completely simulate the infection and immune states of the hepatitis C virus in a primate, so if the genome of the GB virus B is used as a framework, and a functional gene of the hepatitis C virus replaces a corresponding functional region of the GB virus B to construct an HCV/GBV-B chimeric virus, the method is an ideal scheme for researching the immunity of the hepatitis C virus by using an infection model. However, because of the difficulty in replacing the complete functional protein gene of hepatitis c virus into the GB virus B genome and the uncertainty and risk of whether the constructed chimeric virus containing complete functional protein can infect small primates, the current studies using HCV/GBV-B chimeric virus have been found only in replacing the 5' -NCR, p7 or HVR1 of hepatitis c virus of smaller gene segments into the GB virus B genome, and these chimeric viruses containing only short gene segments of hepatitis c virus can only be used to study the functions of these specific gene segments, and cannot show the interaction between hepatitis c virus and host, and cannot fully mimic the immune response of hepatitis c virus in primates. The above protocol for the replacement of 5 '-NCR, p7 or HVR1 of hepatitis C virus into the GB virus B genome is described in Rijnbrand, R.et al, academic GB virus B with 5' nonvariable RNA sequence from a hepatitis C virus genome in tamarins Hepatologys.41, 986-994(2005), Takikawa, S.et al, functional languages of GB virus B p13 protein: development of a recombinant GB virus B hepatitis virus with a p7 protein. Proc Natl Acad Sci USA 103, 3345. sub.3350 (2006), Haqshennas, G., Dong, X., Net, H., Torresi, J. & Gowans, E.J.A. ceramic GB virus B encoding the hepatitis C virus hypervariable region 1 is induction of infection in vivo.J.Gen.Virol.88, 895. sub.902 (2007) and Griffin, S.et al.Chimerac virus B genes induction of infection C virus p7 area induction of infection in vivo.J.908, 2008. sub.915 (49).
Disclosure of Invention
The technical problem to be solved by the invention is to provide a chimeric virus of complete envelope protein of hepatitis C virus and GB virus B, which can infect marmoset monkeys, simulate the infection and immune state of the hepatitis C virus in primates, and carry out immune prevention and treatment research and vaccine evaluation on the hepatitis C virus.
The chimeric virus is formed by sequentially connecting a 5 'end non-coding region and a core protein gene sequence of GB virus B, a complete envelope protein gene sequence of hepatitis C virus, a non-structural protein and a 3' end non-coding region sequence of GB virus B.
The gene structure and the construction mode of the chimeric virus of the complete envelope protein of the hepatitis C virus and the GB virus B are shown in figure 1. Wherein the GB virus B whole genome consists of a 5 'end non-coding region (5' -NCR), a core protein gene (core), a complete envelope protein gene (E1E2p13), non-structural proteins 2 to 5B (NS2-NS3-NS4A-NS4B-NS5A-NS5B) gene and a 3 'end non-coding region (3' -NCR). The dotted line represents the replacement of the corresponding region of GB virus B by the complete envelope protein of hepatitis C virus (E1E2p7) gene (shown in shaded area in the figure), and the resulting chimeric virus complete genome of complete envelope protein of hepatitis C virus and GB virus B consists of the noncoding region (5 ' -NCR) and core protein gene (core) of GB virus B5 ', the complete envelope protein gene of hepatitis C virus (E1E2p7), the nonstructural proteins 2 to 5B (NS2-NS3-NS4A-NS4B-NS5A-NS5B) and the noncoding region (3 ' -NCR) of GB virus B.
The complete envelope protein gene sequence of the hepatitis C virus can be complete envelope protein gene sequences of hepatitis C viruses of different genotypes, and can also be complete envelope protein gene sequences of hepatitis C viruses of different independent sources of the same genotype. The complete envelope protein gene sequence of the hepatitis C virus of the present invention is preferably the complete envelope protein gene sequence of the hepatitis C virus of genotype 1 b. The complete envelope protein gene sequence of 1b genotype hepatitis C virus has 63.2% of nucleotide sequence homology with existing genotypes 1a, 1b, 2a, 2b, 3, 4, 5 and 6 hepatitis C virus, and can represent the complete envelope protein of all genotypes of hepatitis C virus.
When the complete envelope protein gene sequence of the hepatitis C virus is the complete envelope protein gene sequence of the 1b genotype hepatitis C virus, the sequence of the chimeric virus is shown as SEQ ID NO: 1, wherein the 1-913 site is a 5 'end non-coding region and a core protein gene sequence of the GB virus B, the 914-2767 site is a complete envelope protein gene sequence of the hepatitis C virus, and the 2768-9525 site is a non-structural protein and a 3' end non-coding region sequence of the GB virus B.
The chimeric virus of the present invention can be obtained according to a preparation method commonly used in the art, and the method recommended by the present inventors is as follows:
(1) extracting RNA from a 1b type hepatitis C virus serum sample, carrying out RT-PCR reverse transcription to obtain cDNA, carrying out nested PCR amplification on a complete structural protein gene fragment of the hepatitis C virus, and connecting the complete structural protein gene fragment with a pMD-20T vector to obtain a plasmid containing the complete structural protein gene fragment of the hepatitis C virus; wherein the primers for nested PCR amplification are as follows:
the sequence of the upstream outer primer is SEQ ID NO: 2;
the sequence of the downstream outer primer is SEQ ID NO: 3;
the sequence of the upstream inner primer is SEQ ID NO: 4;
the sequence of the downstream inner primer is SEQ ID NO: 5;
(2) taking a plasmid of a GB virus B whole genome, and amplifying a NotI-core at the upstream and a NS2-AflII at the downstream of a complete envelope protein gene of the GB virus B by a PCR method; taking the plasmid obtained in the step (1), and amplifying a complete envelope protein gene segment of the hepatitis C virus by adopting a PCR method; wherein,
the upstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 6;
the downstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 7;
the sequence of the upstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 8;
the sequence of the downstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 9;
the upstream primer sequence for amplifying the fragment of the GB virus B NS2-AflII gene is SEQ ID NO: 10;
the sequence of a downstream primer for amplifying a fragment of the GB virus B NS2-AflII gene is shown as SEQ ID NO: 11;
respectively carrying out electrophoresis on the amplified PCR products and carrying out gel recovery on target bands, and connecting the obtained three sections of DNA by using an overlappingPCR method according to the sequence of the complete envelope protein gene fragment of the hepatitis C virus, the NS2-AflII gene fragment of the GB virus B and the NotI-core gene fragment of the GB virus B to obtain a recombinant DNA fragment;
carrying out double enzyme digestion on the plasmid of the GB virus B whole genome by using NotI and AflII, recovering and removing a DNA fragment containing the complete envelope protein gene fragment of the GB virus B between two sites of the NotI and the AflII, and connecting the DNA fragment with the recombinant DNA fragment to obtain a chimeric whole genome plasmid;
(3) the whole genome sequence of the resulting chimeric whole gene plasmid was cleaved at the SacI site, and RNA of the chimeric virus was synthesized by transcription with T7RNA polymerase using this as a template.
The chimeric virus of the complete envelope protein of the hepatitis C virus and the GB virus B, which is established by the invention, is a fragment of 1854bp long fragment of the complete envelope protein of the hepatitis C virus, compared with the previous report of constructing the chimeric virus by replacing a fragment of GBV-B shorter fragment, is a larger breakthrough, and the complete envelope protein is directly involved in the interaction with host cells to induce an organism to generate neutralizing antibodies and specific cell immunity, and is a main protective antigen.
Drawings
FIG. 1 is a schematic diagram of the gene structure and construction method of a chimeric virus of hepatitis C virus complete envelope protein and GB virus B.
FIG. 2 is a histogram of changes in viral load in serum after intrahepatic injection of chimeric viral RNA in marmoset monkeys; where black squares represent chimeric viruses and white squares represent GB virus B.
FIG. 3 is a histogram of changes in viral load in serum after intravenous inoculation of serum from a primary chimeric virus infected marmoset monkey to a second generation marmoset monkey; where black squares represent chimeric viruses and white squares represent GB virus B.
FIG. 4 shows the electrophoresis chart of RT-nested PCR identification of chimeric virus RNA in primary and passaged marmoset monkey serum.
Detailed Description
The invention is described in further detail below with reference to specific embodiments and with reference to the following drawings. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example 1 preparation of chimeric Virus of the complete envelope protein of hepatitis C Virus and GB Virus B
(1) Preparation of complete structural protein Gene of hepatitis C Virus (including complete envelope protein Gene fragment)
(a) Viral RNA in a hepatitis C virus type 1b serum sample was extracted from 200. mu.l serum using a Roche nucleic acid extraction kit according to the protocol and dissolved in 50. mu.l of precipitation buffer.
(b) Designing and synthesizing an upstream outer primer, a downstream outer primer, an upstream inner primer and a downstream inner primer for amplifying a hepatitis C virus 1b type complete structural protein gene; reverse transcribing the extracted hepatitis C virus RNA into eDNA by using an RT-PCR method; the complete structural protein gene segment of the hepatitis C virus is amplified in two rounds by a nested PCR method. Connecting the amplified complete structural protein gene of the hepatitis C virus with a pMD-20T vector, and transforming with TOP10 competent cells to obtain a plasmid containing the complete structural protein gene segment of the hepatitis C virus.
Wherein, the primers for amplifying the hepatitis C virus type 1b complete structural protein gene are as follows:
the sequence of the upstream outer primer is SEQ ID NO: 2;
the sequence of the downstream outer primer is SEQ ID NO: 3;
the sequence of the upstream inner primer is SEQ ID NO: 4;
the sequence of the downstream inner primer is SEQ ID NO: 5, and (c) a sequence shown in (5).
Wherein, the extracted RNA is reversely transcribed into eDNA, and the reaction system and conditions are as follows (TaKaRa):
the nested PCR method amplifies the complete structural protein gene segment of the hepatitis C virus in two rounds, and the reaction system and conditions are as follows (TaKaRa):
(2) construction of chimeric whole genome plasmid of hepatitis C virus complete envelope protein and GB virus B
(a) Taking a plasmid of a GB virus B complete genome (named pGBB, the gifts of the United states Jens Bukh doctor; the addresses: NIH, NIAID, LID, Hepatitis Virus Section, Building 7, rom 201, 7 Center Dr. MSC 0740, Bethesda, MD 20892. adult. 0740. fax (301)402-0524. E-mail: jbukhniaid. NIH. gov), and amplifying two gene fragments of NotI-core at the upstream and NS2-AflII at the downstream of the complete envelope protein gene of the GB virus B by a PCR method; and (2) taking the plasmid obtained in the step (1), and amplifying the complete envelope protein gene segment of the hepatitis C virus by adopting a PCR method. The primer design meets the requirement of overlapping PCR. Wherein,
the upstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 6;
the downstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 7;
the sequence of the upstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 8;
the sequence of the downstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 9;
the upstream primer sequence for amplifying the NS2-AflII gene fragment of the GB virus B is SEQ ID NO: 10;
the sequence of a downstream primer for amplifying the NS2-AflII gene fragment of the GB virus B is SEQ ID NO: 11, and (c) the sequence shown in fig. 11.
Respectively amplifying the three fragments by using a common PCR method, wherein an amplification system and reaction conditions are respectively as follows:
amplifying the NotI-core gene fragment (TaKaRa) of GB virus B:
amplifying the complete envelope protein gene segment of the hepatitis C virus:
amplifying NS2-AflII gene segment of GB virus B:
the amplified PCR product was electrophoresed and the band of interest was recovered from the gel.
(b) And connecting the recovered three sections of DNA by using an Overlapping PCR method to obtain a recombinant DNA fragment. Firstly, connecting the complete envelope protein gene segment of the hepatitis C virus with the NS2-AflII gene segment of the GB virus B, wherein the reaction system and conditions are as follows:
the target band (hepatitis C virus complete envelope protein and NS2-AflII connecting fragment of GB virus B) obtained by connection is connected with NotI-core gene fragment of GB virus B, and the reaction system and conditions are as follows:
a recombinant DNA fragment is obtained.
(c) And carrying out double enzyme digestion on the plasmid of the GB virus B whole genome by using NotI and AflII, recovering and removing a DNA fragment containing the complete envelope protein gene fragment of the GB virus B between two sites of the NotI and the AflII, and connecting the DNA fragment with the recombinant DNA fragment to obtain the chimeric whole genome plasmid.
(d) After sequencing, SEQ ID NO: 1 is a complete envelope protein of hepatitis C virus and GB virus B chimeric whole genome sequence, full length 9525bp, wherein, the 1-913 bit is the 5 'end non-coding region and core protein gene sequence of GB virus B, the 914-2767 bit is the complete envelope protein gene sequence of 1B genotype hepatitis C virus, the 2768-9525 bit is the non-structural protein and 3' end non-coding region sequence of GB virus B.
(3) Preparation of chimeric virus RNA of hepatitis C virus complete envelope protein and GB virus B
The whole genome sequence of the obtained chimeric whole gene plasmid was cut at the SacI site, and the chimeric virus RNA was synthesized by RNA polymerase transcription using the SacI site as a template and a T7 transcription kit according to the instructions under the guidance of the T7 promoter.
Example 2 evaluation of infectivity of chimeric viruses in marmosets
Evaluation criteria for infectivity of chimeric viruses: the bacterial strain has infectivity: virus replication, virus load is detected in marmoset serum and correct verification is carried out; ② no infectivity: the virus did not replicate and no viral load was detected in marmoset sera.
The method for infecting marmosets may be intrahepatic injection of chimeric virus and intravenous injection of primary marmoset serum containing chimeric virus. The specific infection procedures and evaluations were as follows:
1. determination of viral load in serum of intrahepatic injection chimeric virus RNA and marmoset monkey
(1) Intrahepatic injection of chimeric viral RNA
Healthy adult experimental animals marmosets (ALT, AST and other liver enzyme activities are normal, GB virus B is negative) (about 300-. Respectively establishing:
firstly, infecting marmoset monkey experimental group by chimeric virus;
② the positive control group of marmoset monkey is infected by GB virus B;
③ marmoset negative control group without virus infection.
(2) Determination of viral load in marmoset sera
(a) Upstream and downstream primers and probes are designed according to the gene sequence of the 5' end non-coding region of the GB virus B.
The upstream primer of the non-coding region of the amplification GB virus B5' end is SEQ ID NO: 12;
the downstream primer of the non-coding region of the amplification GB virus B5' end is SEQ ID NO: 13, and (c) a sequence set forth in (c);
the probe for amplifying the non-coding region at the end of GB virus B5' is SEQ ID NO: 14, or a sequence shown in fig. 14.
(b) After the marmoset monkey is inoculated, 0.5-1ml of blood is collected in femoral vein every 1-2 weeks. Serum was isolated and viral RNA was extracted from 100. mu.l serum using Roche nucleic acid extraction kit according to the protocol and dissolved in 50. mu.l of Elution buffer. Then, the extracted viral RNA is reverse transcribed into cDNA by RT-PCR. The upstream and downstream primers and probes for amplifying the 5' non-coding region of the GB virus B are used for determining the in vivo replication condition of the virus by a quantitative PCR method.
The extracted RNA is reversely transcribed into cDNA, and the reaction system and conditions are as follows (TaKaRa):
the quantitative PCR reaction system and conditions were as follows:
the results are shown in FIG. 2. As can be seen from FIG. 2, the hepatitis C virus complete envelope protein and the GB virus B chimeric virus infected marmoset monkey experimental group and the GB virus B positive control group both have typical virus replication and clearance conditions. No virus was detected in the negative control sera. The results prove that the complete envelope protein of the hepatitis C virus and the RNA of the GB virus B chimeric virus can be packaged and replicated in the liver, and the chimeric virus has infectivity in a marmoset monkey.
2. Serum inoculation infection and passage of primary chimeric virus infected marmoset monkey
The marmoset monkey serum containing chimeric virus and GB virus B is respectively subcultured, and the virus inoculation amount is 104copies, serum was intravenously injected into other healthy marmosets, and negative controls were made, and a passaged experimental group of three groups of marmosets in step 1(1) of example 2 was established accordingly. The method is the same as that of step 1(2) in example 2, and comprises collecting blood from femoral vein every 1-2 weeks by 0.5-1ml, and determining virus replication in vivo by Quantitative PCR (QPCR).
The results are shown in FIG. 3. FIG. 3 shows that typical virus replication and clearance conditions appear in the chimeric virus passage infection experimental group of hepatitis C virus complete envelope protein and GB virus B and the GB virus B positive control passage infection group. No virus was detected in the negative control sera. The results prove that the chimeric virus of the complete envelope protein of the hepatitis C virus and the GB virus B can be subjected to subculture infection.
3. Identification of chimeric virus in serum of primary and passage chimeric virus infected marmoset monkey
To further prove that the viruses detected by quantitative PCR in steps 1 and 2 of the above example 2 are chimeric viruses with GB virus B backbone, but not GB virus B whole viruses, primers were designed according to E1 sequence in the complete envelope protein gene sequence of hepatitis C virus and E1 sequence of GB virus B for nested PCR (RT-nested) identification.
(1) The primers were designed based on the E1 sequence in the complete envelope protein gene sequence of hepatitis C virus.
The upstream outer primer for identifying the chimeric virus is SEQ ID NO: 15, or a sequence shown in seq id no;
the downstream outer primer for identifying the chimeric virus is SEQ ID NO: 16;
the upstream inner primer for identifying the chimeric virus is SEQ ID NO: 17;
the downstream inner primer for identifying the chimeric virus is SEQ ID NO: 18, or a sequence shown in fig. 18.
(2) Primers were designed based on the E1 sequence of GB virus B.
The upstream outer primer for identifying the GB virus B is SEQ ID NO: 19;
the downstream outer primer for identifying the GB virus B is SEQ ID NO: 20;
the upstream inner primer for identifying the GB virus B is SEQ ID NO: 21;
the downstream inner primer for identifying the GB virus B is SEQ ID NO: 22, or a sequence shown in fig. 22.
(3) The virus genome extracted from the serum of the primary and passage chimeric virus marmoset infection model is used as a template, and primers for identifying the chimeric virus are used for reverse transcription and nested PCR amplification, and the reaction system and conditions are the same as those in example 1. The results are shown in figure 4, wherein the 2, 3 and 4 pore canals are the chimeric virus model identification results of the complete envelope protein of the primary hepatitis C virus and the GB virus B, and the 5, 6 and 7 pore canals are the chimeric virus model identification results of the complete envelope protein of the passage hepatitis C virus and the GB virus B. The size of the electrophoresis band is 165bp, the amplified product is correct after cloning and sequencing, and the virus detected in the serum of the primary and passage hepatitis C virus complete envelope protein and the GB virus B chimeric virus marmoset infection model is determined to be the chimeric virus of the hepatitis C virus complete envelope protein and the GB virus B.
(4) The virus genome extracted from primary and passage GB virus B marmoset monkey infection model serum is used as a template, and primers for identifying GB virus B are used for reverse transcription and nested PCR amplification, and the reaction system and conditions are the same as those in example 1. The result is shown in figure 4, electrophoresis pore canals are 8-11, the size of an electrophoresis band is 217bp, and the amplified product is correct after cloning and sequencing, so that the virus detected in the serum of the primary and passage GB virus B marmoset infection model is determined to be GB virus B.
Claims (1)
1. A method for constructing chimeric virus RNA of hepatitis C virus complete envelope protein and GB virus B, which comprises the following steps:
(1) extracting RNA from a 1b type hepatitis C virus serum sample, carrying out RT-PCR reverse transcription to obtain cDNA, carrying out nested PCR amplification on a complete structural protein gene fragment of the hepatitis C virus, and connecting the complete structural protein gene fragment with a pMD-20T vector to obtain a plasmid containing the complete structural protein gene fragment of the hepatitis C virus; wherein the primers for nested PCR amplification are as follows:
the sequence of the upstream outer primer is SEQ ID NO: 2;
the sequence of the downstream outer primer is SEQ ID NO: 3;
the sequence of the upstream inner primer is SEQ ID NO: 4;
the sequence of the downstream inner primer is SEQ ID NO: 5;
(2) taking a plasmid of a GB virus B whole genome, and amplifying a NotI-core at the upstream and a NS2-AflII at the downstream of a complete envelope protein gene of the GB virus B by a PCR method; taking the plasmid obtained in the step (1), and amplifying a complete envelope protein gene segment of the hepatitis C virus by adopting a PCR method; wherein,
the upstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 6;
the downstream primer sequence for amplifying the NotI-core gene fragment of the GB virus B is SEQ ID NO: 7;
the sequence of the upstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 8;
the sequence of the downstream primer for amplifying the complete envelope protein gene segment of the hepatitis C virus is SEQ ID NO: 9;
the upstream primer sequence for amplifying the NS2-AflII gene fragment of the GB virus B is SEQ ID NO: 10;
the sequence of a downstream primer for amplifying the NS2-AflII gene fragment of the GB virus B is SEQ ID NO: 11;
respectively carrying out electrophoresis on the amplified PCR products and carrying out gel recovery on target bands, and connecting the obtained three sections of DNA by using an overlappingPCR method according to the sequence of the NotI-core gene fragment of the GB virus B, the complete envelope protein gene fragment of the hepatitis C virus and the NS2-AflII gene fragment of the GB virus B to obtain a recombinant DNA fragment;
carrying out double enzyme digestion on the plasmid of the GB virus B whole genome by using NotI and AflII, recovering and removing a DNA fragment containing the complete envelope protein gene fragment of the GB virus B between two sites of the NotI and the AflII, and connecting the DNA fragment with the recombinant DNA fragment to obtain a chimeric whole genome plasmid;
(3) the whole genome sequence of the obtained chimeric whole gene plasmid was cleaved at the SacI site, and RNA of the chimeric virus was synthesized by transcription with T7RNA polymerase using this as a template.
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| US7244585B1 (en) * | 1999-06-04 | 2007-07-17 | The Board Of Regents Of The University Of Texas System | 3′ Sequence of the GB virus B (GBV-B) genome |
| WO2010008010A1 (en) * | 2008-07-15 | 2010-01-21 | 株式会社先端生命科学研究所 | Hcv/gbv-b chimeric virus |
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| US7244585B1 (en) * | 1999-06-04 | 2007-07-17 | The Board Of Regents Of The University Of Texas System | 3′ Sequence of the GB virus B (GBV-B) genome |
| WO2004005498A1 (en) * | 2002-07-03 | 2004-01-15 | Board Of Regents, The University Of Texas System | Chimeric gb virus b (gbv-b) |
| WO2010008010A1 (en) * | 2008-07-15 | 2010-01-21 | 株式会社先端生命科学研究所 | Hcv/gbv-b chimeric virus |
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| 李婷婷等.F－050 丙型肝炎病毒结构蛋白的嵌合病毒狨猴感染模型.《中国输血杂志》.2010,第23卷(第s1期),167. * |
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