CN109913424B

CN109913424B - Human liver cancer cell line containing hepatitis E virus replicon, application and construction method

Info

Publication number: CN109913424B
Application number: CN201910245093.7A
Authority: CN
Inventors: 黄耀伟; 徐令东
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2020-10-27
Anticipated expiration: 2039-03-28
Also published as: CN109913424A

Abstract

The invention discloses a human liver cancer cell line containing a hepatitis E virus replicon, and an application and a construction method thereof. The human liver cancer cell line is named as human liver cancer cell Huh7-EZ, and the preservation number is CCTCC NO: C2018193. according to the invention, through molecular cloning and reverse genetic technology, the genome of the virus is modified, EGFP/zeocin gene is substituted for the gene of encoding capsid protein of HEV, and bleomycin is added for screening, so that a Huh7 cell line supporting continuous replication of HEV replicon is obtained and named as human liver cancer cell Huh7-EZ, and the cell has important significance for researching HEV pathogenic mechanism and excavating novel anti-virus or screening novel anti-virus medicine, and provides a novel model for HEV research.

Description

Human liver cancer cell line containing hepatitis E virus replicon, application and construction method

Technical Field

The invention relates to the technical field of biomedicine, in particular to a human liver cancer cell line containing a hepatitis E virus replicon, and an application and a construction method thereof.

Background

Hepatitis E Virus (HEV) is the causative agent of Hepatitis E, which is one of the major causes of acute Hepatitis at present. The target population for hepatitis E virus is young adults 15-40 years of age. The clinical symptoms are typical acute and viral hepatitis symptoms, including jaundice, general debilitation, inappetence, nausea, abdominal pain, fever, hepatomegaly, and the like; hepatitis without jaundice has also been reported.

The disease was first developed in 1955 in new delhi in india, and HEV is now prevalent in most parts of asia, africa and latin america. According to the reports of epidemic and sporadic diseases, it is estimated that 20 million people live in the region where hepatitis E is prevalent. A common feature in all endemic areas is that drinking water is contaminated and viruses infect people or animals via the fecal oral route. Hepatitis e is an important public health disease in many developing countries with poor public health conditions due to its specific route of transmission. But there are also sporadic and mass-sending cases in many developed industrialized countries including japan, the united states, and europe. The mortality rate of hepatitis E in the population is 0.2-1%. But unlike other viral hepatitis, it has a unique clinical feature that the mortality rate of pregnant women infected with HEV is as high as 30%. While chronic infection of HEV can occur in immunosuppressed patients.

Mammalian HEV is mainly divided into 4 genotypes, the infected subjects of genotype 1 and genotype 2 are limited to human, genotype 3 and genotype 4 are zoonosis, and animals including pigs, deer and the like are hosts of HEV.

At present, HEV is the main pathogenic cause of acute hepatitis, and because the virus lacks an effective cell culture system at present, the research and development of a medicament for treating HEV are difficult, and no good medicament can be used for treating HEV.

HEV is a single positive stranded RNA virus. The genome of HEV comprises 3 open reading frames, ORF1 encodes a non-structural protein, ORF2 encodes a capsid protein, and ORF3 encodes a small multifunctional protein.

The non-structural protein of HEV is encoded by ORF1, which begins at the 5' end of the non-coding region, and ORF1 encodes a polypeptide chain of 1693 amino acids, which is involved in viral replication and protein processing. ORF1 contains several functional regions including the methyltransferase (Met) region 5' of the cap of the viral RNA genome, the "Y" region of unknown function, the papain-like cysteine protease (PCP) region, the hypervariable region (HVR) in a proline-rich region, the "X" region of unknown function next to the PCP region, the helicase (Hel) region, and the RNA-dependent RNA polymerase region responsible for viral replication.

ORF2 encodes the capsid protein of HEV, having 660 amino acids, and functions to encapsidate viral nucleic acids.

ORF3 encodes a small protein of 123 amino acids which has recently been found to be translated from a bicistronic subgenome. ORF3 is not essential for replication of the virus particle and recent studies have shown that this protein may be an ion channel protein.

The prevention and control of HEV is a difficult problem in China and even the world at present. The difficulty in controlling HEV is mainly manifested in the following aspects:

(1) because HEV cannot infect cells effectively in vitro, HEV in vitro cell culture lines are lacking;

(2) HEV lacks a small animal model;

(3) no drug specific for treatment of HEV;

(4) there are currently no cell lines in the world that can support the sustained replication of HEV.

The replicon systems of viruses are particularly important for studying the interaction mechanism of viruses with hosts in the absence of cell culture systems and animal models. Taking hepatitis C as an example, researchers successfully screen the medicine for treating hepatitis C by using a hepatitis C replication system, and great economic benefits and social benefits are generated. Therefore, if the HEV replicone cell line is established, the HEV replicone cell line has important significance for screening antiviral drugs for hepatitis E.

The Huh7 cell is a liver cancer cell, is derived from human liver cells, is mainly used for culturing HEV, and is an important material in the research of HEV prevention and control technology.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a human liver cancer cell line containing a hepatitis E virus replicon.

A human liver cancer cell line containing hepatitis E virus replicons is named as human liver cancer cells Huh7-EZ, and the preservation number is CCTCC NO: C2018193.

the invention is realized by the following steps of preparing a Hepatitis E Virus (HEV) replicon: the fused EGFP/zeocin resistance gene is inserted into a Kernow-C1p6 infectious clone sequence, Huh7 cells are transfected, zeocin resistance gene is selected by bleomycin (zeocin), and the cells which are alive carry the zeocin gene, namely, the related sequence of the HEV replicon, and emit green fluorescence due to the EGFP gene. Screening is carried out, and finally, a cell which grows well and has green fluorescence is obtained, is named as a human liver cancer cell Huh7-EZ, is preserved in the China center for type culture Collection at Wuhan university in Wuhan, 2018 and 11 months and 3 days, and has the preservation number of CCTCC NO: C2018193. human hepatoma cells Huh7-EZ are subjected to related detection, expression of HEV non-structural protein Helicase can be detected on the protein level through indirect immunofluorescence, and genome and subgenome of a virus replicon can be detected on the RNA level through Northern blot, so that the replicon can complete the whole life cycle of the virus except packaging. When bleomycin is added into the culture medium and continuously passaged for 13 weeks, the positive rate of the HEV replicon is not reduced, which indicates that the HEV replicon can stably replicate in cells.

The invention also discloses the application of the human liver cancer cell line in serving as an in-vitro cell culture system of the hepatitis E virus.

The invention also discloses application of the human liver cancer cell line in the research of interaction mechanism of hepatitis E virus and host.

The invention also discloses application of the human liver cancer cell line in screening anti-hepatitis E virus drugs.

The invention also discloses a construction method of the human liver cancer cell line containing the hepatitis E virus replicon, which comprises the following steps:

(1) inserting a fluorescent protein gene and a resistance screening gene into a Kernow-C1p6 infectious cloning sequence to obtain a p6-EZ HEV replicon vector;

(2) linearizing a p6-EZ HEV replicon vector, and transcribing in vitro to obtain corresponding RNA;

(3) transfecting Huh7 cells with the RNA obtained by in vitro transcription in the step (2);

(4) after transfection, using corresponding resistance selection agent to carry out resistance selection;

(5) and subcloning the cells subjected to resistance screening, and selecting the subclones with corresponding fluorescence signals as the human liver cancer cell lines containing the hepatitis E virus replicon.

The fluorescent protein gene is an EGFP gene, and the resistance screening gene is a zeocin resistance gene. The resistance screening agent is an antibiotic zeocin; in the resistance selection, zeocin as an antibiotic was used at a final concentration of 250ng/ml, and the medium containing zeocin was changed every two days for 2 weeks of continuous selection.

According to the construction method, the fluorescent protein gene and the resistance screening gene in the step (1) are inserted between the sites of restriction enzymes AflII and PmlI of the Kernow-C1p6 infectious cloning sequence.

According to the invention, through molecular cloning and reverse genetics technology, the genome of a virus is modified, EGFP/zeocin gene (fusion gene of green fluorescent protein gene and bleomycin resistance gene) is replaced by the gene of encoding capsid protein of HEV, and bleomycin is added for screening to obtain a Huh7 cell line supporting the continuous replication of HEV replicon, named as human hepatoma cell Huh7-EZ, which has important significance for researching HEV pathogenesis, excavating novel antiviral drugs or screening novel antiviral drugs, and a novel model is provided for HEV research.

Drawings

FIG. 1 is a schematic diagram of the construction of the p6-EZ HEV replicon vector in example 1.

FIG. 2 is a gel electrophoresis test result of the RNA products of p6-EZ HEV replicon in vitro transcription in example 2, wherein lane p6 is a negative control group using original Kernow-C1p6 infectious clone as a template, and lane p6-EZ is a test group using p6-EZ HEV replicon vector as a template.

FIG. 3 is a graph showing the results of Northern Blot analysis of p6-EZ HEV replicon genome and subgenome in human hepatoma cells Huh7-EZ, wherein lanes Huh7-EZ are detection groups and Huh7 is a negative control group using ordinary Huh7 cells (the same below).

FIG. 4 is a graph showing the immunofluorescence assay results for HEV nonstructural proteins in human hepatoma cells Huh7-EZ of example 4.

FIG. 5 is a graph showing the results of the HEV replicon stability assay in human hepatoma cells Huh7-EZ of example 5.

FIG. 6 is a graph showing the results of testing human hepatoma cells Huh7-EZ as an antiviral drug screening model in example 6, using interferon as an example, wherein A is the result of fluorescence microscope observation and B is the result of fluorescence intensity statistical analysis.

Detailed Description

Example 1

Establishment of p6-EZ HEV replicon vectors.

The p6-EZ HEV replicon vector construction flow is shown in FIG. 1: a p 6-EZHHEV replicon vector is constructed by taking Kernow-C1p6(HEV genotype 3; GenBank accession number: JQ679013) infectious clone (hereinafter referred to as HEV infectious clone) as a framework.

The EGFP/zeocin (EZ) gene of interest (hereinafter abbreviated as EZ, the sequence of which is shown in SEQ ID No. 1) is fused 5 'to the 3' end of ORF1 by using a fusion PCR method, and the EGFP/zeocin (EZ) gene is replaced on the cDNA infectious clone of HEV through two restriction enzyme sites of AflII and PmlI.

(1) By PCR, using primers:

the upstream primer AflII 594-F:

5’-GATGTCTCTTAAGGGTTTCTGGAAGAAGCATTC-3’，

downstream primer AflII 594-R/EGFPzeocin:

5’-CCCTTGCTCACCATGGTGATCCCATGGGCGATGC-3’，

the nt 4765-5358 fragment of HEV ORF1 was amplified from cDNA of an HEV infectious clone.

(2) By PCR, using primers:

upstream primer ORF 2-EZ:

5’-gcccatgggatcaccATGGTGAGCAAGGGCGAGGA-3’，

the downstream primer PmlI-EZ-R:

5’-CACCACGTGAATCAGTCCTGCTCCTCGGCCACGAA-3’，

the EGFP/zeocin (EZ) gene was amplified from pTracer-SV40(Invitrogen) vector.

(3) The products obtained by the two PCR are purified by using a Kit GeneJET Gel Extraction Kit (Thermo), then fusion PCR is carried out by using an upstream primer AflII-594-F and a downstream primer PmlI-EZ-R, the fused PCR product is cut by using AflII and PmlI, the fused fragment is inserted into the HEV infectious clone, and meanwhile, the original fragment of the AflII-PmlI region of the infectious clone is deleted. Obtaining the p6-EZ HEV replicon vector, wherein the nucleotide sequence is shown as SEQ ID No. 2.

Example 2

Establishment of hepatitis E virus replicon Huh7 cell line.

(1) In vitro transcription: the plasmid (p6-EZ HEV replicon vector) is linearized by MluI (NEB) restriction enzyme, 1 mu g of linearized p6-EZ HEV replicon vector is taken as a template, and mMESSAGE mMACHINE T7kit (Ambion) in vitro transcription kit is used for incubation at 37 ℃ for 90min, so as to obtain RNA transcribed by the p6-EZ HEV replicon vector. The transcription product was detected by electrophoresis, and the result is shown in FIG. 2, indicating successful transcription.

(2) Transfection: mu.l of DMRIE-C (Invitrogen) was added to 700. mu.l of Opti-mem serum-free medium, mixed well using a vortexer and allowed to react for 30min at room temperature. To Opti-mem was added 10. mu.l of the RNA product of the in vitro transcribed p 6-EZHHEV replicon vector obtained in step (1). And 5 min. Opti-mem was then added to Huh7 cell culture medium and after 5h the cells were changed to complete medium (DMEM high-sugar medium containing 10% fetal bovine serum).

(3) Selection of cells with zeocin resistance: adding a resistance screening agent to the medium: the antibiotic zeocin (Thermo-Fisher) was used at a final concentration of 250 ng/ml. Complete medium containing the antibiotic zeocin was changed every two days and screening was continued for 2 weeks. The screened cells were subjected to cell counting, after which the cells were diluted and the diluted cells were transferred to a 96-well plate for subcloning. After subcloning, one of the Huh7 subclones grows well and has green fluorescence, the cell is named as a human hepatoma cell Huh7-EZ, and is preserved in the China center for type culture Collection, China university of Wuhan, 2018, 11 months and 3 days, with the preservation number of CCTCC NO: C2018193.

as the GFP reporter gene is inserted into the genome of the p6-EZ HEV replicon vector, cells carrying GFP fluorescence are screened (as shown in figure 4), and the success of screening the human hepatoma cells containing the hepatitis E virus replicon Huh7-EZ is demonstrated.

Example 3

Northern Blot detection.

Trizol (thermo) extracts total RNA of human liver cancer cells Huh 7-EZ. Northern blot analysis was performed using the reagents and conditions specified in the Northern Max kit (Ambion). A nucleic acid dye containing formaldehyde was added to 10. mu.g of total RNA, denatured at 65 ℃ for 15 minutes, and subjected to RNA nucleic acid electrophoresis using a 2% agarose formaldehyde gel. RNA was transferred to a nylon membrane by capillary blotting, then UV-crosslinked to the nylon membrane, and prehybridized in a hybridization solution (northern Max; Ambion) at 68 ℃ for 1 hour. After hybridization, a probe labeled with digoxin at the 3' end specific to HEV was added to the hybridization solution: 5'-CACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATG CCGTTCTTCT-3', at 37 ℃ overnight. The membranes were washed and the probes detected with Bright Star BioDetect kit. The full-length genome (8166bp) and subgenome (2819bp) of the HEV replicon exist in the HEV replicone cell line through northern blot detection (as shown in figure 3), which indicates that the HEV replicone is consistent with the fact that the virus replicates in the cell. Sustained replication of HEV RNA replicon was verified at the RNA level in Huh 7-EZ.

Example 4

And (4) indirect immunofluorescence detection.

Fixing the cells: adding 4% paraformaldehyde into human liver cancer cell Huh7-EZ to completely cover the cell, and fixing at 37 deg.C for 20 min; adding the prepared PBS containing 1% BSA into the cells to completely cover the cells, and standing at 37 ℃ for 1 h; rabbit polypeptide antibodies for detecting HEV nonstructural protein Helicase (antibody preparation reference NairVP, et al (2016) endogenous recombinant plasmid Stress Induced Synthesis of novelvirral Factor media infection Replication of Genotype-1Hepatitis E virus. PLoS vaccines 12(4): e1005521.) were diluted 1: 500, added to the cells, and allowed to stand at 37 ℃ for 1 h; diluting fluorescent secondary antibody (Thermo, product number A11034) according to a ratio of 1: 1000, and keeping away from light at 37 ℃ for 1 h; dyeing with DAPI, and reacting at room temperature for 5 min; an aqueous solution of 50% glycerol was added dropwise. Expression of HEV non-structural protein Helicase can be detected by indirect immunofluorescence (as shown in figure 4), which indicates that the p6-EZ HEV replicon has viral non-structural protein expression in human hepatoma cells Huh7-EZ of the hepatitis E virus replicon, and the p6-EZ HEV replicon is verified to be continuously replicated in the cells at the protein level.

Example 5

HEV replicon cell line stability assay.

The HEV replicon cell line was serially subcultured with the addition of the antibiotic zeocin. The proportion of HEV replicon positive cells was measured weekly by flow cytometry. The results are shown in FIG. 5: it was found that the p6-EZ HEV replicon in human hepatoma cell Huh7-EZ of hepatitis E virus replicon can be stably subcultured.

Example 6

And (4) detecting the interferon sensitivity.

Peg-IFN-alpha 2a is added into human hepatoma cells Huh7-EZ to detect whether the replication of p6-EZ HEV in the cells is inhibited or not. The strength of the inhibition effect of the Peg-IFN-alpha 2a on the HEV replicon is judged by detecting the change of the fluorescence intensity of the cells through a flow cytometry analyzer. Referring to the previous literature, 6 concentration gradients (125IU, 250IU, 500IU, 1000IU, 2000IU) were selected, including the control group (no added Peg-IFN-. alpha.2a). The results are shown in fig. 6, observed by fluorescence microscopy in the experiment: with the increase of the concentration of the Peg-IFN-alpha 2a, the fluorescence intensity of the human liver cancer cell Huh7-EZ gradually decreases, and the higher the concentration of the Peg-IFN-alpha 2a is, the stronger the inhibition effect on the HEV replicon is.

Sequence listing

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<120> human liver cancer cell line containing hepatitis E virus replicon, application and construction method

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gcggtaaccc cgatcagaac ctcgggactt tacaagcctt cccgccgtcc tgccagatta 3960

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ccgttcaggc caccacatgt gagttgtatg agttggttga ggccatggtg gagaagggtc 4320

aggacgggtc agccgtctta gagctagatc tctgcaatcg tgatgtctcg cgcatcacat 4380

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ccattgagaa agaaatactg gccctgctcc cgcctaatgt cttttatggc gatgcttatg 4560

aggagtcagt gcttgctgcc gctgtgtcag gggcggggtc atgcatggta tttgaaaatg 4620

acttttcgga gtttgatagc acccagaaca acttctctct cggccttgag tgtgtggtta 4680

tggaggagtg cggcatgcct caatggttaa ttaggttgta tcacctggta cggtcagcct 4740

ggattctgca ggcgccaaag gagtctctta agggtttctg gaagaagcat tctggtgagc 4800

ccggtaccct tctttggaac accgtttgga acatggcaat catagcacat tgctacgagt 4860

tccgtgactt tcgtgttgct gcctttaagg gtgatgattc ggtggtcctc tgtagcgact 4920

accggcagag ccgcaatgcg gcagctttga ttgctggctg tgggcttaaa ttgaaggttg 4980

actatcgccc cattgggctg tatgctgggg tggtggtggc ccctggcttg gggacactgc 5040

ctgatgtggt gcgttttgct ggtcggctgt ccgaaaagaa ttggggcccc ggcccggaac 5100

gtgctgagca gctacgtctt gctgtttgtg atttccttcg agggttgacg aacgttgcgc 5160

aggtctgtgt tgatgttgtg tcccgtgtct atggagttag ccccgggctg gtacataacc 5220

ttattggcat gttgcagacc attgccgatg gcaaggccca ctttacagag actattaaac 5280

ctgttcttga tcttacaaat tccatcatac agcgggtaga atgaataaca tgtttgttgc 5340

atcgcccatg ggatcaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 5400

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 5460

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 5520

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 5580

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 5640

caggagcgcaccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 5700

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 5760

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 5820

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 5880

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 5940

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 6000

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 6060

gacgagctgt acaagtccgc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 6120

gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 6180

gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 6240

caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 6300

gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 6360

gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 6420

gtgcacttcg tggccgagga gcaggactga ttcacgtggt gctattctgc gtcggcagta 6480

taatttgtcc acgtccccgc tcacgtcatc tgttgcttcg ggtaccaatt tggttctcta 6540

cgctgccccg ctaaatcccc tcttgcccct ccaggatggc accaacaccc atatcatggc 6600

tactgaggca tccaactatg ctcagtaccg ggtcgttcga gctacgatcc gctaccgccc 6660

gctggtgccg aatgctgttg gtggttatgc tatttctatt tctttttggc ctcaaactac 6720

aactacccct acttctgttg atatgaattc tattacttcc actgatgtta ggattttggt 6780

ccagcccggt attgcctccg agttagtcat ccctagtgag cgccttcatt atcgcaatca 6840

aggctggcgc tctgttgaga ccacaggtgt ggctgaggag gaggctacct ccggtctggt 6900

aatgctttgc attcatggct ctcctgttaa ctcttatact aatacacctt acactggtgc 6960

gttggggctc cttgattttg cactagagct tgaattcagg aatttgacac ccgggaacac 7020

caacacccgt gtttcccggt ataccagcac agcccgtcat cggttgcgtc gcggtgctga 7080

tgggaccgct gagcttacta ccacagcagc cacacgattt atgaaggatc tgcatttcac 7140

tggcactaat ggcgttggtg aggtgggtcg cggtatcgcc ctgacactgt tcaatcttgc 7200

tgatacgctt ctaggtggtt taccgacaga attgatttcg tcggctgggg gtcagttgtt 7260

ctactcccgc cctgttgtct cggccaatgg cgagccgaca gtgaagttat acacatctgt 7320

ggagaatgcg cagcaagaca agggcattac catcccacac gatatagatt tgggtgactc 7380

ccgtgtggtt attcaggatt atgataatca gcacgagcaa gaccgaccca cgccgtcacc 7440

tgccccctca cgccctttct cagtccttcg cgctaacgat gttttgtggc tctccctcac 7500

tgccgctgag tacgatcagg ctacgtatgg gtcgtctacc aaccctatgt atgtctctga 7560

tacagttacc tttgtcaatg tggccactgg tgctcaggct gttgcccgct ctcttgattg 7620

gtctaaagtt actttggatg gtcgccccct tactaccatt cagcagtatt ctaagacatt 7680

ttatgttctc ccgctccgcg ggaagctgtc cttttgggag gctggcacaa ctagggccgg 7740

ctacccatat aactataaca ccactgctag tgatcaaatt ctgattgaga atgcggccgg 7800

ccatcgtgtc gctatctcca cctacactac cagcctgggt gccggccctg cctcgatctc 7860

cgcggtgggt gtattagccc cacactcggc ccttgctgtt cttgaggaca ctgttgatta 7920

ccctgctcgt gctcacactt ttgatgattt ctgcccggag tgtcgtaccc taggtttgca 7980

gggttgtgca ttccagtcca ctattgctga gcttcagcgc cttaaaacgg aggtaggcaa 8040

aacccgggag tcttaattaa ttccttccgt gcccccttcg cagtcttcct tttggcttta 8100

tttcttattt ctgctttccg cgctccctgg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 8160

aaaaaa 8166

<210>3

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gatgtctctt aagggtttct ggaagaagca ttc 33

<210>4

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cccttgctca ccatggtgat cccatgggcg atgc 34

<210>5

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gcccatggga tcaccatggt gagcaagggc gagga 35

<210>6

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

caccacgtga atcagtcctg ctcctcggcc acgaa 35

<210>7

<211>59

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttct 59

Claims

1. A human liver cancer cell line containing a hepatitis E virus replicon is named as human liver cancer cell Huh7-EZ, and the preservation number is CCTCC NO: C2018193.

2. use of the human liver cancer cell line of claim 1 as an in vitro cell culture system for hepatitis E virus.

3. Use of the human liver cancer cell line of claim 1 in the study of the interaction mechanism of hepatitis E virus with a host for non-disease diagnosis or treatment purposes.

4. The use of the human liver cancer cell line of claim 1 in screening anti-hepatitis E virus drugs for non-disease diagnosis or treatment purposes.