CN102154226B - A chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B - Google Patents

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

A chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B

technical field

The present invention relates to the field of virus, composition and its preparation or purification, in particular to the field of virus modified by introducing foreign gene material.

Background technique

Hepatitis C virus (HCV) is one of the main pathogenic factors of acute/chronic hepatitis transmitted by blood. The prevalence of HCV infection varies greatly around the world, especially in developing countries. Worldwide, approximately 170 million people are currently infected with the hepatitis C virus. 50-85% of hepatitis C virus infected patients develop into chronic hepatitis, 10-20% of them further develop into complex chronic liver diseases such as liver cirrhosis, and 1-5% develop into liver cancer after 20 or 30 years. However, there is still a lack of very effective therapeutic drugs for hepatitis C virus infection at present, and what is more serious is that there is no hepatitis C preventive vaccine in the world so far. Until today, the research on hepatitis C has not made breakthrough progress. The key factor is that because hepatitis C virus can only infect humans and chimpanzees, there is a lack of ideal small primate models for hepatitis C virus infection, which greatly limits Research on the immune control of hepatitis C virus.

Hepatitis C virus is a virus belonging to the Hepavirus genus of the Flaviviridae family. Its genome consists of structural (core, envelope) and nonstructural (protease, helicase, enzyme cofactor) proteins and non-coding regions. 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, and is the main protective antigen. In recent years, the successful cell culture of the whole genome infectious clone of hepatitis C virus has made great progress in the cell culture of hepatitis C virus. However, naturally infected (wild) strains cannot be cultured in cells, especially there is no effective small primate infection model to simulate real HCV infection, which still greatly limits the research on HCV 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 pseudotype viruses are mainly used for research on the interaction between hepatitis C virus protein and cells, the determination of neutralizing antibodies, and gene regulation. Since none of these skeleton viruses have hepatotropic properties, they cannot be used in the research on the immune prevention and treatment of hepatitis C vaccine.

GB virus B (GBV-B) is a virus that is closest to hepatitis C virus and belongs to the Hepavirus genus of the Flaviviridae family. Unlike hepatitis C virus, GB virus B replicates in the small primate marmosets common marmoset (Callithrix jacchuejacchus) and lesser marmoset (Tamarin, Saguinus spp) and causes acute or chronic hepatitis. However, GB virus B is still different from hepatitis C virus, and cannot completely simulate the infection and immune status of hepatitis C virus in primates. Therefore, if the GB virus B genome is used as the backbone, the function of hepatitis C virus The gene replaces the corresponding functional region of GB virus B to construct the HCV/GBV-B chimeric virus, which is an ideal solution to study the immunity of hepatitis C virus by using the infection model. However, there are certain difficulties in replacing the complete functional protein gene of hepatitis C virus into the GB virus B genome, and there are uncertainties and risks in whether the constructed chimeric virus with complete functional protein can infect small primates Therefore, the current research using HCV/GBV-B chimeric virus has only been found in the replacement of the 5'-NCR, p7 or HVR1 of the hepatitis C virus with smaller gene segments into the GB virus B genome, which only contains C Chimeric viruses with very short gene segments of hepatitis virus can only be used to study the function of these specific gene segments, and cannot show the interaction between hepatitis C virus and the host, and cannot completely simulate the interaction of hepatitis C virus in primates immune response. For the above scheme of replacing 5'-NCR, p7 or HVR1 of hepatitis C virus into GB virus B genome, see Rijnbrand, R. et al. Achimeric GB virus B with 5'nontranslated RNA sequence from hepatitis C virus cause shepatitis in tamarins .Hepatology.41, 986-994(2005), Takikawa, S.et al.Functional analyzes 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 3350(2006), Haqshenas, G., Dong, X., Netter, H., Torresi, J.&Gowans, E.J.A chimeric GB virus B encodingthe hepatitis C virus hypervariable region 1 is infectious in vivo.J.Gen.Virol.88 , 895-902 (2007). And Griffin, S. et al. Chimeric GB virus B genomes containing hepatitis C virus p7 are infectious in vivo. J. Hepatol. 49, 908-915 (2008) and other literature reports.

Contents of the invention

The technical problem to be solved by the present invention is to provide a chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B, which can infect marmosets, and simulate the infection and immune status of hepatitis C virus in primates , research and vaccine evaluation of hepatitis C virus immune prevention and control.

A chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B, the chimeric virus consists of the 5' non-coding region of GB virus B and the core protein gene sequence, and the complete envelope protein gene of hepatitis C virus sequence and the non-structural protein of GB virus B and the sequence of the 3' end non-coding region are sequentially connected to form.

The gene structure and construction mode of the chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B according to the present invention are shown in FIG. 1 . Among them, the whole genome of GB virus B consists of 5' non-coding region (5'-NCR), core protein gene (core), complete envelope protein gene (E1E2p13), nonstructural protein 2 to 5B (NS2-NS3-NS4A-NS4B -NS5A-NS5B) gene and 3' non-coding region (3'-NCR). The dotted line represents the replacement of the corresponding region of GB virus B with the complete envelope protein (E1E2p7) gene of hepatitis C virus (indicated by the shaded part in the figure), and the whole genome of chimeric virus obtained from the complete envelope protein of hepatitis C virus and GB virus B is composed of GB virus B 5' non-coding region (5'-NCR) and core protein gene (core), hepatitis C virus envelope protein gene (E1E2p7), GB virus B nonstructural protein 2 to 5B (NS2-NS3-NS4A -NS4B-NS5A-NS5B) gene and 3' non-coding region (3'-NCR).

The complete envelope protein gene sequence of the above-mentioned hepatitis C virus of the present invention can be the complete envelope protein gene sequence of different genotypes of hepatitis C virus, and can also be the complete envelope of hepatitis C virus from different independent sources of the same genotype protein gene sequence. The complete envelope protein gene sequence of the above-mentioned hepatitis C virus of the present invention is preferably the complete envelope protein gene sequence of 1b genotype hepatitis C virus. The nucleotide sequence homology between the complete envelope protein gene sequence of HCV genotype 1b and existing HCV genotypes 1a, 1b, 2a, 2b, 3, 4, 5, and 6 is 63.2%, can represent the complete envelope protein of all genotypes of hepatitis C virus.

Chimeric virus of the present invention, when the complete envelope protein gene sequence of described hepatitis C virus is the complete envelope protein gene sequence of 1b genotype hepatitis C virus, its sequence is shown in SEQ ID NO:1 Among them, positions 1-913 are the 5' non-coding region and core protein gene sequence of GB virus B, positions 914-2767 are the complete envelope protein gene sequence of hepatitis C virus, positions 2768-9525 are GB virus B non-structural proteins and 3' non-coding region sequences.

The chimeric virus of the present invention can be obtained according to the commonly used preparation methods in the art, and the method recommended by the inventor is as follows:

(1) Extract RNA from type 1b hepatitis C virus serum samples and reverse transcribe it into cDNA by RT-PCR, then amplify the complete structural protein gene fragment of hepatitis C virus by nested PCR and connect it with pMD-20T carrier to obtain A plasmid containing the complete structural protein gene fragment of hepatitis C virus; wherein the primers for the nested PCR amplification are as follows:

The upstream outer primer sequence is the sequence shown in SEQ ID NO: 2;

The downstream outer primer sequence is the sequence shown in SEQ ID NO: 3;

The upstream internal primer sequence is the sequence shown in SEQ ID NO: 4;

The downstream internal primer sequence is the sequence shown in SEQ ID NO: 5;

(2) Get the plasmid of the whole genome of GB virus B, adopt the PCR method to amplify the NotI-core upstream of the complete envelope protein gene of GB virus B and the downstream NS2-AflII two gene fragments therefrom; Get the resulting plasmid of step (1) , using the PCR method to amplify the complete envelope protein gene fragment of hepatitis C virus therefrom; wherein,

The upstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 6;

The downstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 7;

The upstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 8;

The downstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 9;

The upstream primer sequence for amplifying the GB virus B NS2-AflII gene fragment is the sequence shown in SEQ ID NO: 10;

The downstream primer sequence for amplifying the GB virus B NS2-AflII gene fragment is the sequence shown in SEQ ID NO: 11;

The amplified PCR products were electrophoresed and the target bands were recovered from the gel. The obtained three segments of DNA were divided into the complete envelope protein gene fragment of hepatitis C virus and the NS2-AflII gene fragment of GB virus B by the method of OverlappingPCR. Sequential connection with the NotI-core gene fragment of GB virus B to obtain a recombinant DNA fragment;

Take another GB virus B whole genome plasmid and use NotI and AflII to perform double enzyme digestion, recover and remove the DNA fragment containing the complete envelope protein gene fragment of GB virus B between the NotI and AflII sites, and connect to the recombinant DNA fragment , to obtain a chimeric whole genome plasmid;

(3) The full genome sequence of the gained chimeric full-gene plasmid is cut at the SacI site, and with it as a template, the RNA of the chimeric virus is transcribed by T7 RNA polymerase.

The chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B established by the present invention is used to replace the fragment of the complete envelope protein of GB virus B with a 1854bp long fragment of the complete envelope protein of hepatitis C virus, compared with the previous replacement The report on the construction of chimeric viruses with shorter fragments of GBV-B is a major breakthrough, and the complete envelope protein directly intervenes in the interaction with host cells, inducing the body to produce neutralizing antibodies and specific cellular immunity, which is the main protective Therefore, after the chimeric virus infects marmosets, it can truly reflect the immune response of hepatitis C virus, which can be used to screen the protective epitope of the complete envelope protein of hepatitis C virus and better understand hepatitis C Viral infection, pathology, host immune response, and preclinical research and evaluation of vaccines and therapeutic antibodies.

Description of drawings

Figure 1 is a schematic diagram of the gene structure and construction method of the chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B.

Figure 2 is a histogram of changes in serum viral load after intrahepatic injection of chimeric virus RNA in marmoset monkeys; wherein, black squares represent chimeric viruses, and white squares represent GB virus B.

Figure 3 is a histogram of viral load changes in the serum of marmosets infected with the primary chimeric virus after intravenous inoculation with the second generation of marmosets; black squares represent chimeric viruses, and white squares represent GB virus B.

Accompanying drawing 4 is the electrophoresis diagram of RT-nested PCR identification of chimeric virus RNA in primary and passage marmoset serum.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Embodiment 1, prepare the chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B

(1) Prepare the complete structural protein gene of hepatitis C virus (which includes the complete envelope protein gene fragment)

(a) Extract viral RNA from HCV type 1b serum samples from 200 μl serum with the Roche Nucleic Acid Extraction Kit according to the instructions, and dissolve in 50 μl Elution buffer.

(b) Design and synthesize the upstream outer primers, downstream outer primers, upstream inner primers and downstream inner primers for amplifying the complete structural protein gene of hepatitis C virus type 1b; reverse the extracted hepatitis C virus RNA by RT-PCR It was recorded as eDNA; the complete structural protein gene fragment of hepatitis C virus was amplified in two rounds by nested PCR. The amplified complete structural protein gene of the hepatitis C virus is connected with the pMD-20T vector, transformed with TOP10 competent cells, and the plasmid containing the complete structural protein gene fragment of the hepatitis C virus is obtained.

Wherein, the primers for amplifying the complete structural protein gene of hepatitis C virus type 1b are as follows:

The upstream outer primer sequence is the sequence shown in SEQ ID NO: 2;

The downstream outer primer sequence is the sequence shown in SEQ ID NO: 3;

The upstream internal primer sequence is the sequence shown in SEQ ID NO: 4;

The downstream internal primer sequence is the sequence shown in SEQ ID NO:5.

Among them, the extracted RNA is reverse-transcribed into eDNA, and the reaction system and conditions are as follows (TaKaRa):

The nested PCR method is divided into two rounds to amplify the complete structural protein gene fragment of hepatitis C virus. The reaction system and conditions are as follows (TaKaRa):

(2) Construction of a chimeric full-genome plasmid of the complete envelope protein of hepatitis C virus and GB virus B

(a) Take the plasmid of the whole genome of GB virus B (named pGBB, donated by Dr. Jens Bukh, USA. Address: NIH, NIAID, LID, Hepatitis Viruses Section, Building 7, Room 201, 7 Center Dr. MSC 0740, Bethesda, MD 20892-0740. Fax: (301) 402-0524. E-mail: jbukhniaid.nih.gov), using the PCR method to amplify the NotI-core upstream of the complete envelope protein gene of GB virus B and the downstream NS2-AflII two A gene fragment; the plasmid obtained in step (1) is taken, and a PCR method is used to amplify the complete envelope protein gene fragment of hepatitis C virus therefrom. Primer design meets the requirements of overlapping PCR. in,

The upstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 6;

The downstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 7;

The upstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 8;

The downstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 9;

The upstream primer sequence for amplifying the NS2-AflII gene fragment of GB virus B is the sequence shown in SEQ ID NO: 10;

The downstream primer sequence for amplifying the NS2-AflII gene fragment of GB virus B is the sequence shown in SEQ ID NO: 11.

The above three fragments were respectively amplified by ordinary PCR method, and the amplification system and reaction conditions were as follows:

Amplify the NotI-core gene fragment (TaKaRa) of GB virus B:

Amplification of the complete envelope protein gene fragment of hepatitis C virus:

Amplify the NS2-AflII gene fragment of GB virus B:

The amplified PCR products were electrophoresed and the target bands were recovered from the gel.

(b) Ligate the recovered three pieces of DNA by the method of Overlapping PCR to obtain a recombinant DNA fragment. First, the complete envelope protein gene fragment of hepatitis C virus is connected with the NS2-AflII gene fragment of GB virus B, and the reaction system and conditions are as follows:

The target band obtained by connection (the complete envelope protein of hepatitis C virus and the NS2-AflII connection fragment of GB virus B) is connected with the NotI-core gene fragment of GB virus B again, and the reaction system and conditions are as follows:

A recombinant DNA fragment is then obtained.

(c) Take the plasmid of the complete genome of GB virus B and carry out double enzyme digestion with NotI and AflII, reclaim and remove the DNA fragment containing the complete envelope protein gene fragment of GB virus B between the two sites of NotI and AflII, and recombine with the The DNA fragments were ligated to obtain a chimeric whole genome plasmid.

(d) After sequencing, SEQ ID NO: 1 shows the chimeric complete genome sequence of the complete envelope protein of hepatitis C virus and GB virus B, the full length is 9525bp, and positions 1-913 are the 5' end of GB virus B Non-coding region and core protein gene sequence, 914-2767 is the complete envelope protein gene sequence of genotype 1b hepatitis C virus, 2768-9525 is the non-structural protein and 3' end non-coding region sequence of GB virus B.

(3) Prepare the chimeric virus RNA of the complete envelope protein of hepatitis C virus and GB virus B

Cut the whole genome sequence of the obtained chimeric whole gene plasmid with SacI site, and use it as a template, use the T7 transcription kit to operate according to the instructions, under the guidance of the T7 promoter, transcribe and synthesize the chimeric virus RNA by RNA polymerase.

Example 2. Infectivity evaluation of chimeric virus in marmoset

Evaluation criteria for chimeric virus infectivity: ① Infectious: virus replicates, and the viral load is detected in marmoset serum and verified to be correct; ② Non-infectious: virus does not replicate, and viral load cannot be detected in marmoset serum.

The method of infecting marmosets can be intrahepatic injection of chimeric virus and intravenous injection of primary marmoset serum containing chimeric virus to infect marmosets. The specific infection steps and evaluation are as follows:

1. Intrahepatic injection of chimeric viral RNA and determination of viral load in marmoset serum

(1) Intrahepatic injection of chimeric virus RNA

Choose healthy adult marmosets (with normal liver enzyme activity such as ALT, AST, and GB virus B negative) (about 300-400g/monkey), divide them into three groups, expose the liver by surgery, and inject the prepared chimeric virus RNA into the liver (dissolved in phosphate buffer before injection), GB virus B or phosphate buffer, the inoculation amount of chimeric virus and GB virus B was 300 μg/only, and sutured. build respectively:

① Chimeric virus infected marmoset experimental group;

②GB virus B infected marmoset positive control group;

③Negative control group of marmosets without virus infection.

(2) Determination of viral load in marmoset serum

(a) Design upstream and downstream primers and probes based on the 5' UTR gene sequence of GB virus B.

The upstream primer for amplifying the non-coding region of the B5' end of GB virus is the sequence shown in SEQ ID NO: 12;

Amplify the downstream primer of the GB virus B5' end non-coding region as the sequence shown in SEQ ID NO: 13;

The amplified GB virus B5' end non-coding region probe is the sequence shown in SEQ ID NO: 14.

(b) After marmoset inoculation, 0.5-1 ml of blood was collected from the femoral vein every 1-2 weeks. The serum was separated, and viral RNA was extracted from 100 μl serum with Roche nucleic acid extraction kit according to the instructions, and dissolved in 50 μl Elution buffer. Then, the extracted viral RNA was reverse-transcribed into cDNA by RT-PCR. Using the above-mentioned upstream and downstream primers and probes for amplifying the 5' non-coding region of GB virus B, the replication of the virus in vivo was determined by quantitative PCR.

The extracted RNA was reverse-transcribed into cDNA, and the reaction system and conditions were as follows (TaKaRa):

The quantitative PCR reaction system and conditions are as follows:

The results are shown in Figure 2. It can be seen from Figure 2 that typical virus replication and clearance conditions appeared in the marmoset experimental group infected with the intact envelope protein of hepatitis C virus and the GB virus B chimeric virus and the GB virus B positive control group. No virus was detected in the serum of the negative control group. The results proved that the complete envelope protein of hepatitis C virus and the chimeric virus RNA of GB virus B can be packaged and replicated in the liver, and the chimeric virus is infectious in marmosets.

2. Serum inoculation infection and passage of primary chimeric virus infected marmosets

The marmoset serum tested to contain chimeric virus and GB virus B was subcultured and inoculated separately, the amount of virus inoculated was 10 ⁴ copies, and the serum was intravenously injected into other healthy marmoset monkeys, and a negative control was made at the same time, and step 1 of Example 2 was established accordingly ( 1) Passage experiment groups of the three groups of marmosets. 0.5-1 ml of blood was collected from the femoral vein every 1-2 weeks, and quantitative PCR (QPCR) was used to measure the replication of the virus in vivo, and the detection method was the same as step 1 (2) of Example 2.

The results are shown in Figure 3. It can be seen from Figure 3 that the chimeric virus subculture infection experiment group of the complete envelope protein of hepatitis C virus and GB virus B and the GB virus B positive control subculture infection group also showed typical virus replication and clearance conditions. No virus was detected in the serum of the negative control group. The results prove that the chimeric virus of the complete envelope protein of hepatitis C virus and GB virus B can be subcultured and infected.

3. Identification of chimeric virus in the serum of marmoset monkeys infected with primary and passage chimeric virus

In order to further prove that the virus detected by quantitative PCR in steps 1 and 2 of the above-mentioned Example 2 is a chimeric virus with a GB virus B skeleton, rather than a whole virus of GB virus B, according to the complete envelope protein gene sequence of hepatitis C virus Primers were designed for the E1 sequence in and the E1 sequence of GB virus B for nested PCR (RT-nested) identification.

(1) Design primers according to the E1 sequence in the complete envelope protein gene sequence of hepatitis C virus.

The upstream outer primer for identifying the chimeric virus is the sequence shown in SEQ ID NO: 15;

The downstream outer primer for identifying the chimeric virus is the sequence shown in SEQ ID NO: 16;

The upstream internal primer for identifying the chimeric virus is the sequence shown in SEQ ID NO: 17;

The downstream internal primer for identifying the chimeric virus is the sequence shown in SEQ ID NO: 18.

(2) Design primers according to the E1 sequence of GB virus B.

Identify the upstream outer primer of GB virus B as the sequence shown in SEQ ID NO: 19;

Identify the downstream outer primer of GB virus B as the sequence shown in SEQ ID NO: 20;

Identify the upstream inner primer of GB virus B as the sequence shown in SEQ ID NO: 21;

The downstream internal primer for identifying GB virus B is the sequence shown in SEQ ID NO: 22.

(3) Using the virus genome extracted from the serum of the marmoset infection model of the primary and passage chimeric virus as a template, reverse transcription and nested PCR amplification were performed with primers for identifying the chimeric virus, and the reaction system and conditions were the same as in Example 1. The results are shown in Figure 4, in which channels 2, 3, and 4 are the identification results of the chimeric virus model of the primary hepatitis C virus complete envelope protein and GB virus B, and channels 5, 6, and 7 are the complete envelope of the passaged hepatitis C virus Protein and GB virus B chimeric virus model identification results. The size of the electrophoresis band is 165bp, and the amplified product is correct after cloning and sequencing, indicating that the virus detected in the serum of the chimeric virus marmoset infection model of primary and passage hepatitis C virus and GB virus B is indeed type C Chimeric virus of hepatitis virus intact envelope protein and GB virus B.

(4) Using the virus genome extracted from the serum of the primary and passage GB virus B marmoset infection model as a template, carry out reverse transcription and nested PCR amplification with primers for identifying GB virus B, and the reaction system and conditions are the same as in Example 1. The results are shown in Figure 4, as shown in the electrophoresis wells 8-11, the size of the electrophoresis band is 217bp, and the amplified product is correct after cloning and sequencing, indicating that the virus detected in the serum of the primary and passage GB virus B marmoset infection model is indeed GB virus B.

Claims (1)

1. a method for constructing the chimeric viral RNA of hepatitis C virus complete envelope protein and GB virus B, the method is made up of the following steps: (1) Extract RNA from type 1b hepatitis C virus serum samples and reverse transcribe it into cDNA by RT-PCR, then amplify the complete structural protein gene fragment of hepatitis C virus by nested PCR and connect it with pMD-20T carrier to obtain A plasmid containing the complete structural protein gene fragment of hepatitis C virus; wherein the primers for the nested PCR amplification are as follows: The upstream outer primer sequence is the sequence shown in SEQ ID NO: 2; The downstream outer primer sequence is the sequence shown in SEQ ID NO: 3; The upstream internal primer sequence is the sequence shown in SEQ ID NO: 4; The downstream internal primer sequence is the sequence shown in SEQ ID NO: 5; (2) Get the plasmid of the whole genome of GB virus B, adopt the PCR method to amplify the NotI-core upstream of the complete envelope protein gene of GB virus B and the downstream NS2-AflII two gene fragments therefrom; Get the resulting plasmid of step (1) , using the PCR method to amplify the complete envelope protein gene fragment of hepatitis C virus therefrom; wherein, The upstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 6; The downstream primer sequence for amplifying the NotI-core gene fragment of GB virus B is the sequence shown in SEQ ID NO: 7; The upstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 8; The downstream primer sequence for amplifying the complete envelope protein gene fragment of hepatitis C virus is the sequence shown in SEQ ID NO: 9; The upstream primer sequence for amplifying the NS2-AflII gene fragment of GB virus B is the sequence shown in SEQ ID NO: 10; The downstream primer sequence for amplifying the NS2-AflII gene fragment of GB virus B is the sequence shown in SEQ ID NO: 11; The amplified PCR products were electrophoresed and the target bands were recovered from the gel. The obtained three segments of DNA were divided into the NotI-core gene segment of GB virus B and the complete envelope protein gene segment of hepatitis C virus by the method of OverlappingPCR. Sequential connection with the NS2-AflII gene fragment of GB virus B to obtain a recombinant DNA fragment; Take another GB virus B whole genome plasmid and use NotI and AflII to perform double enzyme digestion, recover and remove the DNA fragment containing the complete envelope protein gene fragment of GB virus B between the NotI and AflII sites, and connect to the recombinant DNA fragment , to obtain a chimeric whole genome plasmid; (3) cutting the whole genome sequence of the obtained chimeric whole gene plasmid at the SacI site, and using it as a template to transcribe and synthesize the RNA of the chimeric virus by T7 RNA polymerase.

Images