6xj type hepatitis C virus subgenome replicon and application thereof

Abstract

The invention discloses a 6xj type hepatitis C virus subgenome replicon, the nucleotide sequences of which are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, the 6xj type hepatitis C virus subgenome replicon is transfected into human liver cancer cells, and 6xj type hepatitis C virus subgenome replicon cells capable of efficiently replicating HCV RNA (Ribonucleic Acid) are obtained through screening. According to the 6xj type hepatitis C virus subgenome replicon, the sensitivity of the 6xj type hepatitis C virus subgenome replicon to velpatasvir and Sofosbuvir drugs is detected by utilizing the replicon system, the replicon is proved to be capable of being applied to hepatitis C virus drug screening, and the 6xj type hepatitis C virus subgenome replicon provides an important research tool for research in the fields of HCV drug-resistant site analysis, adaptive mutation functions, HCV pathogenesis, drug screening and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a 6xj type hepatitis C virus subgenomic replicon and an application thereof. Background Art

[0002] Hepatitis C virus (HCV) infection is a serious global health problem caused by hepatitis C virus (HCV). HCV infection is mainly characterized by liver damage, which can cause acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HCV is a highly diverse single-stranded positive-sense RNA virus with a genome of approximately 9,600 bases, containing a single open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs). The ORF encodes a polyprotein precursor that is subsequently cleaved by host and viral proteases into structural proteins (core protein, E1, E2) and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B), which are essential for viral replication and assembly.

[0004] The complexity and diversity of HCV genotypes are also one of the main reasons for its poor treatment prognosis. Currently, 8 HCV genotypes and 95 subtypes have been found. Of particular concern is the prevalence and complexity of HCV genotype 6 (GT6) in Southeast Asia and southern China, which contains many subtypes with high variability. GT6 represents a large proportion of chronic HCV infection in these regions, but this genotype population has largely remained underappreciated in medical research and treatment. In Yunnan Province, China, several new GT6 subtypes have been discovered, including the recently discovered 6xi and 6xj. This high variability poses a major challenge to effective disease control and the development of broad-spectrum direct-acting antivirals (DAAs).

[0005] The advent of pan-genotypic DAAs (i.e., drugs that are effective against multiple HCV genotypes) has revolutionized the treatment of chronic hepatitis C. However, the high mutation rate of HCV and the genetic diversity between subtypes, especially in GT6, pose a persistent challenge to the efficacy of DAAs. Thirty-one GT6 subtypes have been identified, of which eight new subtypes were first reported in China, with four in Yunnan Province alone. This highlights the rapid evolution of GT6 and underscores the urgent need for targeted drug research and development.

[0006] HCV subgenomic replicons are RNA molecules that can replicate autonomously in cells and contain the HCV nonstructural proteins NS3-NS5B, essential UTRs, and a selectable marker gene (e.g., neomycin phosphotransferase, Neo) and have become an indispensable tool in HCV research. These replicons mimic HCV replication in cell culture but do not produce infectious viral particles. They allow researchers to study HCV replication, identify antiviral drug targets, evaluate the efficacy of DAAs, and study resistance mechanisms. Although replicons have been successfully developed for genotypes 1a, 1b, 2a, 3a, 4a, 5a, and 6a, a critical gap remains in the replicon system for the 6xj subtype, which is prevalent and highly variable in China. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a 6xj type hepatitis C virus subgenomic replicon and its application. The nucleotide sequence of the replicon is as shown in SEQ ID NO: 1, which provides an effective tool for hepatitis C virus research and drug development.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] 1. The viral genomic DNA / RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) was used to extract 6xj hepatitis C virus RNA. The RNA was reverse transcribed and used as a template to amplify the NS3-NS5B region sequence of 6xj hepatitis C virus. The subreplicon plasmid pcc1413-6xj with an adaptive NS5A-S232I point mutation (serine at position 232 of the NS5A protein mutated to isoleucine) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The inserted fragment of the plasmid included the T7 promoter, the 5'UTR sequence and part of the Core region sequence of the HCV 2a subtype, the Neo gene, the EMCV gene IRES sequence, the NS3-NS5B region sequence of HCV 6xj, and the 3'UTR region sequence of the HCV 2a subtype;

[0010] 2. Using plasmid pcc1413-6xj as a template, primers puc19_F_6xjNS and puc19_R_6xjNS were used to amplify the complete insert fragment, primers 6xjNS_F_puc19 and 6xjNS_R_puc19 were used to linearize the puc19 vector, and the insert fragment and the linearized puc19 vector were seamlessly cloned using a seamless cloning kit to construct the replicon puc19-HCV-6xj, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0011] The nucleotide sequence of the replicon puc19-HCV-6xj at positions 1-23 is the T7 promoter sequence, the nucleotide sequence at positions 24-418 is the 5′UTR and part of the Core sequence of HCV-2a, the nucleotide sequence at positions 419-1213 is the Neo gene sequence, the nucleotide sequence at positions 1232-1850 is the IRES sequence of EMCV, the nucleotide sequence at positions 1851-7820 is the non-structural gene (NS3-NS5B) sequence of the virus, the nucleotide sequence at positions 7821-8056 is the 3′UTR sequence of HCV-2a, and the nucleotide sequence at positions 8057-18977 is the backbone plasmid pcc1413 sequence;

[0012] 3. Using the replicon puc19-HCV-6xj successfully constructed by sequencing as a template, PCR amplification was performed using mutant primers, and the amplified products were recovered for homologous recombination to obtain 6xj hepatitis C virus subgenomic replicons puc19-HCV-6xj-V28M, puc19-HCV-6xj-A442V, and puc19-HCV-6xj-V28M / A442V, the nucleotide sequences of which are shown in SEQ ID NO: 2-4;

[0013] 4. After the replicon plasmid is sequenced and verified to be correct, the replicon plasmid is linearized and then transcribed in vitro to obtain the corresponding RNA, which is then transfected into human liver cancer Huh7.5.1 cells. By adding G418, a hepatitis C virus replicon human liver cancer cell line that can effectively replicate HCV RNA is obtained.

[0014] Another purpose of the present invention is to use the 6xj type hepatitis C virus subgenomic replicon in screening anti-hepatitis C virus drugs.

[0015] Advantages and technical effects of the present invention:

[0016] The invention is based on the discovery in previous studies that there are HCV 6xj NS5A-V28M and NS5B-A442V mutation sites in HCV relapse populations. The viral genome is modified by molecular cloning and seamless cloning technology to obtain a replicon with an adaptive NS5A-S232I mutation and a replicon with single and double mutations of NS5A-V28M and NS5B-A442V of HCV 6xj. The 6xj hepatitis C virus subgenomic replicon is transfected into human liver cancer Huh7.5.1 cells. After adding G418 for screening for 3-4 weeks, a replicon capable of efficiently replicating HCV is obtained. The invention discloses a 6xj type hepatitis C virus subgenomic replicon model of RNA. The invention utilizes the replicon system to detect its sensitivity to velpatasvir and sofosbuvir drugs, and proves that the replicon can be applied in the screening of hepatitis C virus drugs. The 6xj type hepatitis C virus subgenomic replicon of the invention provides an important research tool for the research in the fields of HCV resistance site analysis, adaptive mutation function, HCV pathogenic mechanism and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of agarose gel electrophoresis of two long fragments in Example 1, where lanes 1-3 correspond to DNA Marker, Puc19 vector, and 6xj insert fragment, two seamlessly cloned long fragments, respectively.

[0018] Figure 2 It is the agarose gel electrophoresis diagram of the replicon plasmid puc19-HCV-6xj, in which M is a DNA marker;

[0019] Figure 3 Agarose gel electrophoresis images of replicon plasmids puc19-HCV-6xj-28M, puc19-HCV-6xj-442V, and puc19-HCV-6xj-28M / 442V, where M is a DNA marker;

[0020] Figure 4 It is the linearized agarose gel electrophoresis diagram of the four replicon plasmids in Example 2, wherein lanes 1-5 correspond to DNA Marker, puc19-HCV-6xj, puc19-HCV-6xj-28M, puc19-HCV-6xj-442V, and puc19-HCV-6xj-28M / 442V, respectively;

[0021] Figure 5The results of real-time fluorescence quantitative PCR detection of HCV RNA loads in four 6xj replicon stably transfected cells in Example 3, in which 6xj is puc19-HCV-6xj cells, 6xj-28M is puc19-HCV-6xj-28M cells, 6xj-442V is puc19-HCV-6xj-442V cells, and 6xj-28M / 442V is puc19-HCV-6xj-28M / 442V cells;

[0022] Figure 6 This is the agarose gel electrophoresis diagram of the target fragment amplified by RT-PCR in four replicon cells of 6xj in Example 4;

[0023] Figure 7 The Sanger sequencing peak diagram of the PCR products of the four replicon cells of 6xj in Example 4 verifies the mutation site detection results;

[0024] Figure 8 This is a graph showing the effects of four subreplicon cells of 6xj in Example 5 on different concentrations of the antiviral drug Velpatasvir;

[0025] Fig. 9 This is a graph showing the effects of the four subreplicon cells of 6xj in Example 6 on different concentrations of the antiviral drug Sofosbuvir. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below by examples and drawings, but the scope of the present invention is not limited to the contents. The methods in the examples are carried out according to conventional operations unless otherwise specified, and the reagents used are conventionally purchased reagents or reagents prepared according to conventional methods unless otherwise specified.

[0027] Example 1: Construction of 6xj-type hepatitis C virus subgenomic replicon

[0028] 1. Amplification of HCV NS3-NS5B

[0029] The plasma containing HCV 6xj subtype virus was used to extract viral RNA using a viral genomic DNA / RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). The specific steps are as follows:

[0030] (1) Preparation of carrier RNA solution: centrifuge lyophilized powder containing 310 μg of carrier RNA to the bottom of the tube, then add 310 μL of RNase-Free ddH2O and shake to completely dissolve to obtain a carrier RNA solution with a final concentration of 1 μg / μL, and store at -20°C;

[0031] (2) Preparation of carrier RNA working solution: 0.22 mL of GB solution plus 6.2 μL of 1 μg / μL carrier RNA solution per sample;

[0032] (3) In a clean 1.5 mL centrifuge tube, add 20 μL of Proteinase K solution using a micropipette;

[0033] (4) After the plasma sample is equilibrated to room temperature, 200 μL is added to the above centrifuge tube;

[0034] (5) Add 200 μL of Carrier RNA working solution to the upper tube and vortex for 15 seconds to mix thoroughly;

[0035] (6) Incubate at 56°C for 15 min and centrifuge to collect the liquid attached to the tube wall and tube cap;

[0036] (7) Take 250 μL of anhydrous ethanol and add it to the upper tube. Flocculent precipitation may appear at this time. Vortex and shake for 15 seconds to mix thoroughly. Incubate at room temperature for 5 minutes. If the ambient temperature is higher than 25°C, the anhydrous ethanol needs to be precooled.

[0037] (8) Quickly separate to collect the liquid attached to the tube wall and tube cover;

[0038] (9) Transfer all the above liquid to the adsorption column CR2, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0039] (10) Take 500 μL of GD buffer (confirm whether a certain amount of anhydrous ethanol has been added before use) and add it to the adsorption column. Centrifuge at 8000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0040] (11) Take 600 μL of PW rinse solution (before use, confirm whether a certain amount of anhydrous ethanol has been added) and add it to the adsorption column. Place it at room temperature for 2 min, centrifuge it at 8000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0041] (12) Repeat step (11);

[0042] (13) Take 500 μL of anhydrous ethanol and add it to the adsorption column. Centrifuge at 8000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0043] (14) Centrifuge at 12000 rpm for 3 min to fully dry the adsorption membrane and discard the waste liquid;

[0044] (15) Place a 1.5 mL RNase-Free centrifuge tube into the adsorption column, open the cap of the adsorption column, and leave it at room temperature for 3 min to allow the adsorption film to completely dry. Add 40 μL RNase-Free ddH2O to the middle part of the adsorption film in mid-air, cover the tube, leave it at room temperature for 5 min, and centrifuge at 12000 rpm for 1 min to elute the RNA into a 1.5 mL EP tube; divide the RNA into smaller pieces according to experimental needs and freeze it at -80°C for later use.

[0045] The viral RNA obtained in step 1 was reverse transcribed and used as a template. The primers shown in Table 1 were used for sequence amplification. The amplified products were sent for testing. The obtained sequences were compared to obtain the complete sequence of 6xj NS3-NS5B. The complete sequence of 6xj NS3-NS5B was sent to Beijing Qingke Biotechnology Co., Ltd. to synthesize a subreplicon plasmid pcc1413-6xj with an adaptive NS5A-S232I point mutation. The inserted fragment of the plasmid included a T7 promoter, a 5'UTR sequence and a part of the Core region sequence of the HCV 2a subtype, a Neo gene, an EMCV gene IRES sequence, a NS3-NS5B region sequence of HCV 6xj, and a 3'UTR region sequence of the HCV 2a subtype.

[0046] Table 1

[0047]

[0048] 3. Using plasmid pcc1413-6xj as a template, primers puc19_F_6xjNS and puc19_R_6xjNS in Table 2 were used to amplify the complete insert fragment, and primers 6xjNS_F_puc19 and 6xjNS_R_puc19 were used to linearize the puc19 vector. The PCR reaction system was shown in Table 3. The PCR reaction conditions were 98°C for 30s; 98°C for 10s, 60°C for 5s, 72°C for 5s / kb, 35 cycles; 72°C for 1min; the results of agarose gel electrophoresis of PCR products were shown in Table 3. Figure 1 , PCR products were recovered by gel recovery to obtain the insert fragment and linearized puc19 vector;

[0049] Table 2

[0050]

[0051]

[0052] Table 3

[0053]

[0054] 4. Use the seamless cloning kit (NEB) to seamlessly clone the insert fragment recovered in step 3 and the linearized puc19 vector. The reaction system is shown in Table 4. Mix gently and then centrifuge instantly. Connect at a constant temperature of 50°C for 2 hours in a PCR instrument. After the connection reaction is completed, take 5 μL of the connection product and add it to 100 μL of DH5α Escherichia coli solution, and ice bath for 30 minutes; heat shock in a 42°C water bath or metal bath for 45 seconds, take it out and put it on ice for 2 minutes; add 895 μL of liquid LB culture medium without ampicillin, and shake it on a shaker at 37°C and 200 rpm for 1h30min; after the end, centrifuge at 4000 rpm for 5 minutes, remove the supernatant, keep 100-200 μL of the culture solution and mix it by blowing, evenly apply it to LB solid culture medium with ampicillin, and incubate the plate in an inverted plate in a constant temperature incubator at 37°C overnight;

[0055] Table 4

[0056]

[0057] After overnight culture, a few monoclonal colonies grew on the plate. Use a 10 μL pipette tip to gently pick the monoclonal colonies and put them into 1 mL of liquid LB medium with ampicillin (ampicillin:LB=1:1000). Pick 10 to 20 monoclonal colonies and shake them at 37°C and 200 rpm for about 3 hours. Take 1 μL of the bacterial solution for verification of bacterial solution PCR. The primers used for bacterial solution PCR reaction are shown in Table 5, the PCR reaction system is shown in Table 6, and the reaction conditions are 98°C for 30s; 98°C for 10s, 60°C for 5s, 72°C for 5s / kb, 35 cycles; 72°C for 1min;

[0058] Table 5

[0059]

[0060] Table 6

[0061]

[0062] The positive clones verified by PCR were expanded and the plasmids were extracted. The detailed steps of plasmid extraction are as follows:

[0063] (1) The bacterial solution with positive verification results was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and 4 mL of bacterial solution was pipetted and mixed evenly, and then evenly added to the sterilized EP tube;

[0064] (2) Centrifuge at 12000 rpm for 1 min and discard the supernatant as much as possible;

[0065] (3) Add 250 μL of P1 solution (check to ensure that RNase A has been added before use) to the EP tube containing the bacterial pellet, and vortex until the bacterial solution is completely suspended;

[0066] (4) Add 250 μL of P2 solution to the EP tube and gently invert it upside down 6-8 times to fully lyse the bacteria;

[0067] (5) Add 350 μL of P3 solution to the EP tube and gently invert it upside down 7 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min.

[0068] (6) Column equilibration: Add 500 μL of BL equilibration solution to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0069] (7) After centrifugation in step (5), collect the supernatant, try not to aspirate the precipitate, add it to the adsorption column CP3 in step (6), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0070] (8) Add 500 μL of PD deproteinized solution to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0071] (9) Add 600 μL of PW rinse solution to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reuse the collection tube;

[0072] (10) Repeat step 9;

[0073] (11) Centrifuge at 12000 rpm for 2 min to remove the residual liquid in the adsorption column CP3. After centrifugation, open the lid and place it in a vent to allow the residual liquid in the adsorption membrane to fully dry.

[0074] (12) Place the adsorption column CP3 in a clean EP tube, drip 50-100 μL of 56°C preheated sterile water into the middle of the adsorption column, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, collect the plasmid solution in the EP tube, and the plasmid verification results are shown in Figure 2 .

[0075] 5. Construction of single-point mutation and double-mutation plasmids for drug-resistant mutations NS5A-V28M and NS5B-A442V

[0076] Using the plasmid extracted in step 4 as a template, point mutation primers were designed. The primer sequences were shown in Table 7. PCR amplification was performed according to the reaction system shown in Table 8. The reaction conditions were: 98°C for 30s; 98°C for 10s, 60°C for 5s, 72°C for 5s / kb, 35 cycles; 72°C for 1min. After amplification, agarose gel electrophoresis was performed. After confirming the target band, DpnI was used for digestion to remove the methylated template plasmid. The reaction system was shown in Table 9. After gently blowing and mixing, the mixture was placed in a 37°C constant temperature incubator for reaction for 1 to 2h. After digestion, the gel was recovered.

[0077] Table 7

[0078]

[0079] Table 8 Point mutation subreplicon plasmid amplification reaction system

[0080]

[0081]

[0082] Table 9 DpnI digestion reaction system

[0083]

[0084] 6. Perform homologous recombination on the gel recovery products to complete the cyclization process of the amplified products. The single-point mutation and double-point mutation reaction systems are shown in Table 10 and Table 11, respectively. The optimal DNA usage for the single-point mutation recombination reaction system is 0.003 pmol (about 0.02 × the number of base pairs of the target plasmid); the optimal DNA usage for the double mutation recombination reaction system is 0.03 pmol (i.e. 0.02 × the number of base pairs of the target plasmid) for the longer fragment and 0.06 pmol (i.e. 0.04 × the number of base pairs of the target plasmid) for the shorter fragment. After adding all the systems to the bottom of the tube, pipette and mix, centrifuge briefly, and place at 37 ° C for 30 min. After the reaction, the reaction tube was immediately placed in an ice bath for 5 minutes, and then the entire 20 μL reaction solution was transformed into 100 uL competent cells, placed on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, and then placed in an ice bath for 2 minutes. Finally, 880 μL LB liquid culture medium was added, and the tube was placed in a shaker at 37°C and 200 rpm for 1 hour and 30 minutes. After the reaction, centrifuged at 4000 rpm for 5 minutes, the supernatant was removed, and 100-200 μL of bacterial solution was retained and mixed by pipetting, and evenly applied to LB solid culture medium with ampicillin, and incubated in an inverted plate in a 37°C constant temperature incubator overnight. Subsequently, single clones were picked according to the above method, and PCR bacterial solution verification and point mutation verification were performed (see Figure 3 ), and obtained the replicon plasmids puc19-HCV-6xj-V28M, puc19-HCV-6xj-A442V, and puc19-HCV-6xj-V28M / A442V;

[0085] Table 10 Single point mutation reaction system

[0086]

[0087] Table 11 Double mutation reaction system

[0088]

[0089]

[0090] Example 2: Construction of 6xj-type hepatitis C virus subgenomic replicon stably transfected cells

[0091] 1. After the four replicon plasmids in Example 1 were sequenced and verified to be correct, the replicon plasmids were linearized using the XhoⅠ restriction enzyme, and the linearized products were detected by electrophoresis. The results were as follows: Figure 4 As shown, it indicates that the linearized plasmid is successful. Take 1 μg of the linearized enzyme digestion product and use the T7 in vitro transcription kit to react in a PCR instrument at 37°C for 4 hours to synthesize the RNA chain; the T7 in vitro transcription reaction system is as follows:

[0092]

[0093] 2. After in vitro transcription, RNA purification is performed;

[0094] (1) Add 80uL RNase-Free ddH2O to the RNA sample after in vitro transcription to make the total system 100uL, add 350uL working solution (the working solution is prepared and used immediately, working solution = RK solution: β-mercaptoethanol = 100:1), mix by inversion, and centrifuge briefly;

[0095] (2) Add 250uL of anhydrous ethanol to the solution in the previous step, mix by inverting, and proceed to the next step immediately;

[0096] (3) Transfer the liquid in step (2) to adsorption column CR2, centrifuge at 12000 rpm for 1 min, discard the waste liquid and reuse the collection tube;

[0097] (4) Add 500uL of RW rinse solution to the adsorption column CR2, let it stand at room temperature for 2 minutes, centrifuge at 12000rpm for 1 minute, discard the waste liquid, and reuse the adsorption column;

[0098] (5) Repeat step (4);

[0099] (6) Centrifuge at 12000 rpm for 5 min to fully remove residual liquid;

[0100] (7) Place the adsorption column CR2 in a new EP tube, add 14-20 μL RNase-Free ddH2O to the middle of the adsorption column, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min;

[0101] (8) Measure the concentration of the collected liquid.

[0102] 3. Transfection: Take 4 μL DMRIE-C and add it to 500 μL Opti-MEM serum-free medium. Vortex gently and spin it off. Add 3 μg of in vitro transcribed RNA purified in step 2 to Opti-MEM. Then, immediately drip the Opti-MEM solution slowly into the well plate. After 5 hours, change the cells to DMEM medium without double antibody and containing 10% FBS.

[0103] 4. Screening of cells with Neo resistance: Add resistance screening agent G418 to DMEM medium containing 1% double antibody and 10% FBS. The final concentration of G418 is 1000 μg / mL. Replace the medium containing G418 every two days. After 4 weeks of continuous screening, no dead cells will appear in the cell culture process. At this time, there will be sporadic cell colonies in the well plate, and the cell colonies will be expanded and cultured. The maintenance concentration of G418 in the expanded culture is 1 / 2 of the screening concentration, that is, 500 μg / mL; four stable cells of 6xj hepatitis C virus subgenomic replicons are obtained, namely Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V.

[0104] Example 3: Real-time fluorescence quantitative PCR detection

[0105] Total RNA from Huh7.5.1 cells, Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V cells was extracted by hand extraction RNAiso Plus (Takara), and then the RNA loading of all transfected replicon cells was detected by probe-based absolute quantitative PCR. The quantitative primers, probes, and reaction system are shown in the following table. The reaction conditions were 45°C for 5 min; 95°C for 10 s; 95°C for 5 s, 58°C for 45 s, and 40 cycles. The results are shown in the following table. Figure 5 As shown, the HCV RNA loads of Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V cells were all above Log10 5(copies / mL) or more, indicating that the four constructed HCV 6xj replicon plasmids were successfully transfected into the cells;

[0106]

[0108] Example 4: RT-PCR detection of mutation sites

[0109] 1. Use RNAiso Plus (Takara) to extract total RNA from Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V transfected cells, and use RNA as a template for one-step RT-PCR amplification. Use reverse transcription primers (6xj28F01 / 6xj28R01 and 6xj442F01 / 6xj442R01) and the reaction system as shown in the table below. The reaction conditions are 50℃30min; 94℃3min; 94℃30s, 50℃30s, 72℃1min / kb, 40 cycles; 72℃7min; the kit used is PrimeScript TM One Step RT-PCR Kit (RR055A), product lengths were 594 bp and 572 bp;

[0110]

[0111] 2. Use the PCR product obtained in step 1 as the peripheral template to amplify the inner PCR. The primers used are 6xj28F02 / 6xj28R02 and 6xj442F02 / 6xj442R02. The reaction system is as shown in the following table. The reaction conditions are 94°C for 5 min; 94°C for 30 s, 50°C for 30 s, 72°C for 1 min / kb, 40 cycles; 72°C for 7 min. The DNA enzyme used is Vazyme's Green Taq Mix enzyme. The product lengths are 466 bp and 462 bp.

[0112]

[0113]

[0114] The point mutation sites of the four transfected cells were verified by RT-PCR, and the fragment sizes were consistent with the target fragment size. The results are as follows Figure 6 As shown, the PCR product of the target band was subjected to Sanger sequencing, and the sequencing result peak was single and complete, as shown in Figure 7 As shown, it was shown that the four constructed HCV 6xj replicon plasmids were successfully transfected into the cells and retained the point mutation sites modified in the early stage.

[0115] Example 5: Detection of the sensitivity of replicon stably transfected cells Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V to velpatasvir

[0116] In a 12-well plate, 1.5-2×10 5 The stably transfected cells Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V were inoculated with 100 μg / mL of RNAiso Plus (Takara) and incubated in a CO2 constant temperature incubator (Thermo, USA) at 37°C and 5% CO2 for 24 h. After the cells adhered to the wall and the cell mass reached 80-90% and was in good condition, they were treated with different concentrations of velpatasvir as shown in the following table. After 72 h of drug treatment, 500 μL of cells were collected per well using RNAiso Plus (Takara), and nucleic acids of cells with different drug concentrations were extracted using the hand-lift method. The nucleic acid concentration was determined using a Tiangen ultraviolet spectrophotometer, and the loading amount was calculated (i.e., the purpose of ensuring a uniform loading amount is to ensure absolute quantification). The loading amount used in this embodiment is 1000 ng, and the kit used is One Step PrimeScript TM RT-PCR Kit (RR064A), and the HCV viral load in the collected cells after the above drugs were detected by real-time fluorescence quantitative method, and the quantitative results were processed by Excel software, and then the graphs were drawn and EC were calculated using GraphpadPrism 9 software. 50 The half-maximal effect concentration (the drug concentration that inhibits 50% of the viral load) was calculated.

[0117] The results are as follows Figure 8 As shown, the effect concentration EC of velpatasvir on four transfected cells: Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V 50 The (nM) values ​​were approximately 234.4nM, 10909nM, 4270nM, and 34400nM, respectively. The results showed that Huh7.5.1-6xj cells showed extremely high sensitivity to the anti-HCV drug velpatasvir, and it was found that mutations at two sites, NS5A-V28M and NS5B-A442V, resulted in stronger resistance of subreplicons to the drug;

[0118]

[0119]

[0120] Example 6: Detection of sensitivity of replicon stably transfected cells Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V to sofosbuvir

[0121] In a 12-well plate, 1.5-2×10 5 The stably transfected cells Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V were inoculated with 100 μg / mL of RNAiso Plus (Takara) and incubated in a CO2 constant temperature incubator (Thermo, USA) at 37°C and 5% CO2 for 24 h. After the cells adhered to the wall and the cell mass reached 80-90% and was in good condition, they were treated with different concentrations of sofosbuvir as shown in the following table. After 72 h of drug treatment, 500 μL of cells were collected per well using RNAiso Plus (Takara), and the nucleic acids of cells with different drug concentrations were extracted by hand extraction. The nucleic acid concentration was determined using Tiangen UV spectrophotometer, and the loading amount was calculated (i.e., the purpose of ensuring a uniform loading amount is to ensure absolute quantification). The loading amount used in this embodiment is 1000 ng, and the kit used is One Step PrimeScript TM RT-PCR Kit (RR064A) was used to detect the HCV viral load in the collected cells after the action of the above drugs by real-time fluorescence quantitative method, and the quantitative results were processed by Excel software. Then, GraphpadPrism 9 software was used to draw charts and calculate EC50 (half-maximal effect concentration, i.e., the drug concentration that inhibits 50% of the viral load).

[0122] The results are as follows Fig. 9As shown, the effect concentration EC50 (nM) values ​​of sofosbuvir on four types of cells, Huh7.5.1-6xj, Huh7.5.1-6xj-V28M, Huh7.5.1-6xj-A442V, and Huh7.5.1-6xj-V28M / A442V, were approximately 159.6 μM, 270.6 μM, 133.4 μM, and 403.9 μM, respectively. The results showed that Huh7.5.1-6xj cells showed high sensitivity to the anti-HCV drug sofosbuvir. The drug resistance caused by single mutations in the two sites NS5A-V28M and NS5B-A442V was not much different from that of the original strain. When the two sites NS5A-V28M and NS5B-A442V mutated at the same time, it led to stronger drug resistance.

[0123]

[0124]

[0125] Therefore, the above-mentioned replicon cells can be used as a favorable tool for subsequent screening of other anti-HCV drugs and discovery of new drug-resistant mutation sites.

Claims

1. A 6xj type hepatitis C virus subgenomic replicon, whose nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

2. Use of the 6xj type hepatitis C virus subgenomic replicon according to claim 1 in screening anti-hepatitis C virus drugs.

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