Cell model capable of supporting hepatitis B virus replication and infection and establishing method
阅读说明：本技术 一种可支持乙肝病毒复制和感染的细胞模型及建立方法 (Cell model capable of supporting hepatitis B virus replication and infection and establishing method ) 是由 谢幼华 宋迎迎 刘晶 于 2021-09-10 设计创作，主要内容包括：本发明提供了一种可支持乙肝病毒复制和感染的细胞模型及建立方法。本发明的发明人在实验中发现BEL-7404细胞具有较高的转染效率,在过表达人牛磺胆酸钠共转运体多肽(hNTCP)和肝细胞核因子(HNF4α)基因后可以较好的支持乙肝病毒的复制和感染,以此构建了可稳定表达hNTCP和HNF4α的7404NT-HNF4α细胞模型,该培养系统可通过在培养基中添加强力霉素(Doxycycline,DOX)来调控HNF4α的表达进而调控HBV的复制和感染,为乙肝病毒的研究提供一种新的细胞模型。(The invention provides a cell model capable of supporting hepatitis B virus replication and infection and an establishment method thereof. The inventor of the invention finds that BEL-7404 cells have higher transfection efficiency in experiments, and can better support the replication and infection of hepatitis B virus after overexpression of human sodium taurocholate cotransporter polypeptide (hNTCP) and hepatocyte nuclear factor (HNF4 alpha) genes, so that a 7404NT-HNF4 alpha cell model capable of stably expressing hNTCP and HNF4 alpha is constructed, and the culture system can regulate the expression of HNF4 alpha by adding Doxycycline (DOX) in a culture medium so as to regulate the replication and infection of HBV, thereby providing a new cell model for hepatitis B virus research.)
1. A cell model, wherein the cell model is BEL-7404-hNTCP cells.
2. A method of constructing the cell model of claim 1, the method comprising: through constructing hNTCP slow virus expression system, the BEL-7404cell is transduced, and the BEL-7404-hNTCP cell is obtained through antibiotic screening.
3. A cell prepared based on the cell model of claim 1, wherein the cell is BEL-7404-hNTCP-Tet-on-HNF4 α cell.
4. A method of preparing the cell of claim 3, comprising: BEL-7404-hNTCP cells are infected by using an HNF4 alpha lentivirus expression system, and the BEL-7404-hNTCP-Tet-on-HNF4 alpha cells are obtained through antibiotic screening and monoclonal purification.
5. The method according to claim 4, wherein the HNF4 α lentivirus system is constructed by using the Tet-on expression system, and can be induced and regulated by doxycycline.
6. The method of claim 4, comprising:
s1: constructing hNTCP and HNF4 alpha recombinant lentivirus plasmids;
s2: infecting BEL-7404 cells with hNTCP recombinant lentivirus;
s3: constructing a lentivirus infection system containing HNF4 alpha by using a Tet-on system;
s4: BEL-7404-hNTCP cells are infected by using a lentivirus infection system containing HNF4 alpha obtained from S3, and purified BEL-7404-hNTCP-Tet-on-HNF4 alpha cells are obtained through screening.
7. The cell model of claim 1, the use of the cell of claim 3 in hepatitis B virus research.
8. The use according to claim 7, wherein said use is for studying replication and infection of hepatitis B virus.
9. The use according to claim 7, wherein the cell model or cells are used for screening drugs having inhibitory effect on HBV.
Technical Field
The invention relates to the technical field of cell modification, in particular to application of a 7404NT-HNF4 alpha cell model which is constructed on BEL-7404 cells and highly expresses hNTCP and HNF4 alpha genes in researching hepatitis B virus replication and infection.
Background
Hepatitis B Virus (HBV) is a hepatotropic enveloped virus containing incomplete double-stranded DNA. It is reported that there are at least 2.57 hundred million people infected with HBV worldwide, and chronic infection with HBV can cause clinical diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, which greatly endanger human life health. The existing antiviral treatment (interferon and nucleotide analogs) can only inhibit the replication of the virus to a certain extent, but cannot effectively eliminate the virus, and cannot achieve the aim of curing chronic hepatitis B.
After HBV enters hepatocytes by binding to sodium ion-taurocholate transporting polypeptide (hNTCP) on the surface of human cell membrane, its 3.2kb circular partially double-stranded dna (rcdna) genome, which is wrapped in nucleocapsid, is introduced into nucleus and converted into covalently closed circular dna (cccDNA), followed by transcription of downstream genomic mRNA using cccDNA as a template. Transcription of the virus is regulated by four promoters (Cp, Sp1, Sp2 and Xp) acting in conjunction with enhancers (Enhancer I, EnI) and Enhancer II (Enhancer II, EnII). The core promoter (Cp) plays a key role in the viral life cycle, initiating synthesis of a 3.5kb preC/pregenomic mRNA, encoding e-antigen, core protein and polymerase. Experiments prove that a large number of binding sites of liver-specific and pan-generic transcription factors exist in the promoter region, and the transcription factor HNF4 alpha (hepatocyte nuclear factor 4 alpha) is an important member of a liver-enriched transcription factor family and can participate in regulating and controlling the transcription of Cp, so that the generation of 3.5kb pre/pregenic RNA of HBV is regulated and controlled, and further the regulation and control effect on HBV replication is exerted.
Infection and replication of HBV in vivo has strong hepatophilia and clear species specificity. Although several cellular models have been established to support HBV replication and infection, they all have certain drawbacks. Human primary hepatocytes (PHH) can be infected with HBV in vitro, but are expensive, difficult to obtain and quickly susceptible; heparg cells need to support HBV infection through a long-term differentiation process and have relatively complex culture conditions; after the hNTCP is proved to be a hepatitis B receptor, an HBV infected cell model is constructed on the basis of hepatoma cell lines HepG2 and Huh7, but the two cell transfection efficiencies are low, and the infection effect is not ideal. For the research of HBV, more new cell models are still needed to be found.
BEL-7404 is a liver cancer cell strain, is derived from a 69-year-old Chinese male, is usually used for liver cancer research, and has not seen the application thereof in HBV replication and infection research.
Disclosure of Invention
In view of the above-mentioned problems and/or other problems of the related art, the present invention has been made in view of the above-mentioned and/or other problems, and has been made in an effort to evaluate the application of the 7404NT-HNF4 alpha cell model constructed based on BEL-7404 in HBV studies by in vitro experiments. The in vitro experiment mainly comprises the measurement of cell transfection efficiency, the detection of HBV replication, the construction of a 7404NT-HNF4 alpha cell model capable of stably expressing hNTCP and HNF4 alpha, the infection experiment of HBV and the like. The invention aims to solve the problem that a cell model capable of supporting HBV replication and infection is urgently needed at present, and provides a new application of 7404NT-HNF4 alpha in research of HBV replication and infection.
The technical scheme of the invention is as follows: through constructing hNTCP slow virus expression system, transduction BEL-7404cell, through experimental verification and antibiotic screening BEL-7404-hNTCP cell. Then, an HNF4 alpha lentivirus expression system which can be induced and controlled by Doxycycline (DOX) is constructed by using a Tet-on system, BEL-7404-hNTCP cells constructed in the previous step are infected, and the BEL-7404-hNTCP-Tet-on-HNF4 alpha (named as 7404NT-HNF4 alpha) cells are obtained through antibiotic screening and monoclonal purification. Experimental validation to support HBV replication and infection was then performed.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a cell model in a first aspect, wherein the cell model is BEL-7404-hNTCP cells.
Preferably, the BEL-7404-hNTCP cells include BEL-7404 cells expressing hNTCP.
Preferably, hNTCP is highly expressed in the BEL-7404 cells.
Preferably, said expression comprises mRNA and/or protein expression.
Preferably, hNTCP has Gene ID of 6554; RefSeq NM _ 003049.
In some embodiments of the invention, a 7404NT-HNF4 alpha cell model that can support HBV replication and infection was constructed based on the BEL-7404cell line.
Preferably, the expression of hNTCP and HNF4 alpha genes is enhanced in BEL-7404 cells. Preferably, hNTCP has Gene ID of 6554; RefSeq NM _ 003049.
Preferably, HNF4 α has Gene ID of 3172; RefSeq NM _ 001287182.
In a second aspect, the invention discloses a method for constructing the cell model, which comprises the following steps: through constructing hNTCP slow virus expression system, the BEL-7404cell is transduced, and the BEL-7404-hNTCP cell is obtained through antibiotic screening.
The third aspect of the invention discloses a cell prepared based on the cell model, and the cell is BEL-7404-hNTCP-Tet-on-HNF4 alpha cell.
In a fourth aspect, the invention discloses a method for preparing the above cell, comprising: BEL-7404-hNTCP cells are infected by using an HNF4 alpha lentivirus expression system, and the BEL-7404-hNTCP-Tet-on-HNF4 alpha cells are obtained through antibiotic screening and monoclonal purification.
Preferably, the Tet-on expression system is used for constructing an HNF4 alpha lentivirus system which can be induced and controlled by doxycycline.
Preferably, the method comprises the following steps:
s1: constructing hNTCP and HNF4 alpha recombinant lentivirus plasmids;
s2: infecting BEL-7404 cells with hNTCP recombinant lentivirus;
s3: constructing a lentivirus infection system containing HNF4 alpha by using a Tet-on system;
s4: BEL-7404-hNTCP cells are infected by using a lentivirus infection system containing HNF4 alpha obtained from S3, and purified BEL-7404-hNTCP-Tet-on-HNF4 alpha cells are obtained through screening.
The fifth aspect of the invention discloses a cell prepared based on the cell model, wherein the cell is a 7404NT-HNF4 alpha cell.
Preferably, the 7404NT-HNF4 alpha cells include BEL-7404 cells expressing hNTCP and HNF4 alpha.
Preferably, hNTCP is highly expressed in the 7404NT-HNF4 alpha cell.
Preferably, the expression level of HNF4 alpha is high in the 7404NT-HNF4 alpha cell.
Preferably, said expression comprises mRNA and/or protein expression.
Preferably, hNTCP has Gene ID of 6554; RefSeq NM _ 003049.
Preferably, HNF4 α has Gene ID of 3172; RefSeq NM _ 001287182.
The sixth aspect of the invention discloses the application of the cell model and the cell in hepatitis B virus research.
Preferably, the study is an in vitro study.
Preferably, the study is a non-diagnostic study.
Preferably, the study is a non-therapeutic study.
Preferably, the use is for studying replication and infection of hepatitis B virus.
Preferably, the cell model or the cell is used for screening drugs having inhibitory effect on HBV.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
The invention provides a new application of a 7404NT-HNF4 alpha cell strain as a cell model for HBV replication and infection research. BEL-7404 has the advantages of high transfection efficiency, simple culture conditions and the like, and can support the generation of complete HBV replication and HBV infection after over-expression of hNTCP and HNF4 alpha genes. The expression of HNF4 α can be regulated by addition of DOX in the culture medium, and thus, the transcription, replication, cccDNA formation and HBV infection of HBV in the 7404NT-HNF4 α cell model can also be regulated by DOX.
Compared with the prior art, the invention has the following remarkable advantages and effects:
based on the advantages of higher transfection efficiency of BEL-7404, simpler culture conditions and the like, the invention establishes a 7404NT-HNF4 alpha cell model capable of stably expressing hNTCP and HNF4 alpha and a method thereof based on the key action of hNTCP and HNF4 alpha in the HBV infection replication process. In addition, the invention provides a new application of 7404NT-HNF4 alpha in research of HBV replication and infection.
Drawings
FIG. 1 is a graph showing the results of experiments in which the fluorescence intensity and transfection efficiency after transfection of pCMV-EGFP plasmid in BEL-7404 were observed by fluorescence microscopy and flow cytometry.
FIG. 2 is a schematic diagram showing the result of the experiment that over-expression of HNF4 alpha gene in BEL-7404 by Southern blot can support complete HBV replication.
FIG. 3 is a diagram related to the construction of 7404NT-HNF4 alpha cell line, and the expression levels of hNTCP and HNF4 alpha in cells are detected by real-time quantitative PCR (qPCR) and Western blot.
FIG. 4 is a diagram showing the detection of HBsAg and HBeAg expression levels in cell supernatants by enzyme-linked immunosorbent assay (ELISA) (Southern blot assay for detecting HBV replication in 7404NT-HNF 4. alpha. cells and ELISA for detecting HBV infection in 7404NT-HNF 4. alpha. cells, verifying that the cell model supports HBV replication and infection).
FIG. 5 is a schematic diagram of the construction of hNTCP recombinant lentivirus plasmid.
FIG. 6 is a diagram showing the construction of a HNF4 alpha recombinant lentiviral plasmid constructed using the Tet-on system.
Term(s) for
In the present invention, the term "hNTCP" refers to sodium taurocholate cotransporter polypeptide.
In the present invention, the term "HNF 4 α" refers to hepatocyte nuclear factor 4 α.
In the present invention, the terms "BEL-7404", "BEL-7404 CELL LINE" and "human liver cancer CELL LINE" are used interchangeably.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to fig. 1 to 6 and the embodiments, but the present invention is not limited to the scope of the embodiments.
In the screening process of a cell line capable of supporting HBV replication and infection, the inventor finds that the BEL-7404cell line can better support HBV replication and infection after overexpressing hNTCP and HNF4 alpha genes, has higher transfection efficiency, and can be applied to research on HBV replication and infection mechanisms and screening of drugs with inhibitory action on HBV.
In one embodiment of the present invention, a new use of BEL-7404 in HBV research is provided.
In a preferred embodiment of the invention, a 7404NT-HNF4 alpha cell model which is constructed based on BEL-7404 and can support HBV replication and infection is provided, and the model can be applied to research on HBV replication and infection mechanisms and screening of drugs with inhibitory effect on HBV.
In addition, the invention discloses a new application of the 7404NT-HNF4 alpha cell model in HBV research, and the technicians in the field can develop other cell models taking the 7404NT-HNF4 alpha cell model as a precursor and other applications of the 7404NT-HNF4 alpha cell model acceptable in biological research on the basis of learning the suggestion.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Examples
Information of hNTCP (Gene ID: 6554; RefSeq NM-003049)
Information of HNF4 alpha (Gene ID: 3172; RefSeq NM-001287182)
Example 1
The transfection efficiency after transfection of the pCMV-EGFP plasmid with BEL-7404 was observed by fluorescence microscopy and cell flow experiments:
1. BEL-7404 cells (BEL-7404 cells provided by cell bank/stem cell bank of Chinese academy of sciences, catalog number TCTU 64) suspension (8X 10) were seeded into 6-well plates5One/well), the plates were pre-cultured in an incubator for 12 hours, and the cells grew adherently.
2. Transfection experiments were performed using 4. mu.l of Turbofection transfection reagent (Thermo Fisher Scientific, Waltham, USA) and 2. mu.g of pCMV-EGFP plasmid per well, incubated at 37 ℃ for 48 hours, and the effect of fluorescence expression was observed under a fluorescence microscope, the results of which are shown in FIG. 1A. The pCMV-EGFP plasmid in the step is constructed by the applicant, and the specific method comprises the following steps: the EGFP cDNA sequence was cloned into the pCDN3.1(V79520, Invitrogen) vector using PCR combined with restriction and ligation.
3. Digesting the cells in a pore plate by pancreatin, blowing and uniformly mixing the cells by a pipette to prepare single cell suspension, sucking the single cell suspension into a 1.5ml EP tube, visually observing cell precipitates at the tube bottom of the EP tube, and removing supernatant.
4. 1ml of Cell stabilizing Buffer was added to the Cell pellet, after resuspending the cells, 350g was centrifuged for 5min, and the supernatant was discarded and repeated twice.
5. Resuspending the cells with a Cell stabilizing Buffer, counting the number of viable cells, according to the number of cells 5X 106cells/ml these cell suspensions were aliquoted into new 1.5ml EP tubes at 100 μ l/tube, the samples were transferred to glass tubes for on-machine detection, labeled, followed by flow cytometry for sorting assays, followed by data analysis using FlowJo software. The results of the experiment are shown in FIG. 1B.
The experimental result shows that BEL-7404 cells have higher transfection efficiency and higher fluorescence expression intensity compared with the Huh7 cell line after being transfected with pCMV-EGFP plasmid.
Example 2
The Southern blot experiment detects that the HNF4 alpha gene is over-expressed in BEL-7404 to support the replication of complete HBV, and the specific experiment comprises the following steps:
1. the cell suspension BEL-7404 (1X 10) was inoculated into a 60mm petri dish6One/dish), the dish is placed in an incubator for pre-culture for 12 hours, and cells grow adherently.
Mu.g of pHNF4 α was co-transfected with 4. mu. g p 1.3.3 × HBV replicon plasmid into BEL-7404 cells using 16. mu.l Turbofect transfection reagent, and the control group was transfected with 4. mu.g of the empty plasmid and 4. mu. g p 1.3.3 × HBV replicon plasmid. Extracting HBV DNA in cells after 96h to carry out Southern blot experiment. Among them, pHNF4 α was constructed by cloning HNF4 α cDNA sequence into pcdn3.1(V79520, Invitrogen) vector using PCR in combination with restriction and ligation.
The p1.3 x HBV replicon plasmid is constructed by using HBV strain sequence as template and cloning 1.3 copies of HBV genome into pUC18(Cat.3218, Takara) vector step by using PCR combined with restriction enzyme digestion method. The sequence has been uploaded to the NCBI official website under the sequence number KR232337 (reference: Shen, Z.; Yang, H.; Yang, S.; Wang, W.; Cui, X.; Zhou, X.; Liu, W.; Pan, S.; Liu, Y.; Zhang, J.; Xie, Y.; Liu, J.; Hepatis B viruses per in microorganisms IL-21and IL-33as microorganisms of viral clearence Nat Commun 2017,8, (1),2119.)
2. Extraction of DNA from intracellular HBV core particles
1) Cell lysis: the supernatant was discarded from the transfected cells, washed 2 times with pre-cooled PBS, and the cells were lysed with 400. mu.l lysis buffer (0.5% V/V NP40, 1mM EDTA, 50mM NaCl, 10mM Tris-HCl, pH 7.9) for 15min per 100mM dish.
2) Removal of residual plasmid and cellular genomic DNA: the cell lysate was collected, centrifuged at 14000g for 5min, the supernatant collected and the cell debris discarded. Mu.l of 1M magnesium chloride, 8. mu.l of 10mg/ml DNase I, was added and digested in a water bath at 37 ℃ for 30 min.
3) PEG precipitated virus particles: the digest was centrifuged at 14000g and the supernatant was retained. Mu.l of 0.5M EDTA and 100. mu.l of 35% PEG8000/1.75M sodium chloride were added, mixed well and precipitated overnight at 4 ℃. Centrifuge at 14000g for 10min and discard the supernatant to leave a precipitate.
4) Second removal of residual plasmid and cellular genomic DNA: the pellet was resuspended in 100. mu.l DNase I solution (1. mu.l 1M Tris-HCl, pH7.9, 1. mu.l 10mg/ml DNase I, 1M magnesium chloride, remainder filled with water). Digestion was carried out at 37 ℃ for 30 min.
5) Proteinase K digestion, removal of viral capsid: to the above solution, 300. mu.l of SDS/proteinase K solution was added and digested at 37 ℃ overnight.
6) Phenol/chloroform extraction, precipitation of viral DNA: the mixture was extracted twice with an equal volume of phenol/chloroform, and then 2. mu.l of 20mg/ml glycogen, 1/10 volumes of 3M sodium acetate (pH 5.2) solution and an equal volume of isopropanol were added, mixed well and precipitated at-20 ℃ overnight. Centrifuging at 15000g for 15min, discarding the supernatant, washing the precipitate with 75% ethanol twice and discarding the ethanol, standing until the residual ethanol is volatilized, and dissolving with 20 μ l sterilized distilled water.
Southern blot detection of HBV replication
Agarose electrophoresis: the extracted intracellular HBV DNA was subjected to 1% agarose gel electrophoresis (100V, 1.5 h).
2) Denaturation: the gel after electrophoresis is placed in a fresh denaturing solution (0.5M NaOH and 1.5M NaCl) and is subjected to shake denaturation at room temperature for 1 h.
3) Neutralizing: the denatured solution was discarded, neutralized by pouring a neutralizing solution (1.5M NaCl and 1M Tris-HCl, pH 7.4) and shaken twice at room temperature for 30min each time.
4) Film transfer: using the downward capillary transfer method, the transfer systems are respectively from bottom to top: absorbent paper, Parafilm, 2 layers of 3mm filter paper, nylon membrane, DNA-containing agarose gel, 2 layers of 3mm filter paper, salt bridges immersed in 20 XSSC buffer (3M NaCl and 0.3M sodium citrate) at both ends. And (5) rotating the film for more than 8 hours at normal temperature.
5) DNA crosslinking: and (3) after membrane conversion, taking out the nylon membrane, soaking the nylon membrane in 2 XSSC for 5min, draining off redundant liquid, placing the nylon membrane between two pieces of filter paper, and performing ultraviolet crosslinking for 90 s.
Pre-hybridization: the membrane was placed in a hybridization tube, 5ml of hybridization solution was added, and prehybridization was carried out at 42 ℃ for 30 min.
7) Probe denaturation and hybridization: taking a proper amount of probe, denaturing the probe for 5min in a metal bath at 100 ℃, and then quickly placing the denatured probe on ice for 5 min. Recovering the prehybridization solution, replacing 5ml of fresh hybridization solution, adding the denatured probe, and hybridizing at 42 ℃ for 6-8 h.
8) Washing to remove unbound probe: the wash was first performed 2 times in 2 XSSC at room temperature for 5min each time. Washing with 0.5 XSSC at 68 deg.C for 2 times, each for 15 min;
9) and (3) sealing: diluting 10 × Blocking solution to 1 × Blocking solution with Maleic acid buffer, adding appropriate amount of Blocking solution, and Blocking at 37 deg.C for 30 min.
10) Incubation of the antibody: diluting anti-DIG AP antibody with the 1 Xblocking solution (antibody is diluted according to a ratio of 1: 10000), discarding the blocking solution, replacing with an appropriate amount of antibody incubation solution, and incubating at 37 ℃ for 30 min.
11) Unbound antibody was washed away: the membrane was washed with washing buffer 2 times for 15min each at 37 ℃.
12) Color development: the membrane was equilibrated with a detection buffer for 5min, and diluted with the detection buffer to prepare a1 × CSPD developing solution. And (3) placing the balanced nylon membrane on a Parafilm membrane, enabling the front side of the nylon membrane to face upwards, uniformly dropwise adding a developing solution, covering a layer of Parafilm membrane, removing bubbles and redundant developing solution on the front side of the nylon membrane, keeping the Parafilm membrane away from light, standing the Parafilm membrane at room temperature for 5min, detecting an accumulated signal by using a chemiluminescence detection instrument, and storing a result.
The experimental results are shown in FIG. 2, and the stronger HBV replication can be detected after BEL-7404 is transfected by pHNF4 alpha and HBV replicon plasmid together, and the replication generation is not detected by p1.3 x HBV replicon transfected alone.
Example 3
7404NT-HNF4 alpha cell line is constructed, and the expression level of hNTCP and HNF4 alpha in the cells is detected by real-time quantitative PCR (qPCR) and Western blot
1. Construction of 7404NT-HNF4 alpha cell line: the construction of stable cell lines is carried out step by means of lentivirus infection.
Schematic diagrams of the construction of hNTCP and HNF4 alpha recombinant lentivirus plasmids are shown in FIG. 5 and FIG. 6, respectively. Wherein, FIG. 5 is a HNF4 alpha plasmid map constructed based on Tet-on expression control system. The expression of the system requires the expression of two genes of Tet-on and TRE-HNF4 alpha. The principle is as follows: TRE is a response promoter of Tet-on, and can activate Tet-on expression in the presence of tetracycline (DOX), so that the TRE promoter is activated to start transcription of HNF4 alpha, therefore, a two-step lentivirus infection process is carried out in the cell line construction process, the left picture in FIG. 6 is a Tet-on plasmid expression map used in the first step, and the right picture is a TRE-HNF4 alpha plasmid expression map (FLAG is a tag gene convenient to detect). Both used the same lentiviral expression vector but inserted a different expression gene.
1) Preparing a recombinant lentivirus concentrated solution of hNTCP and HNF4 alpha, and transfecting a lentivirus packaging plasmid psPAX2(Addge, Watertown, USA), pMD2.G (Addge, Watertown, USA) and an hNTCP overexpression plasmid pCDH-hNTCP-Blast or pLVX-Tet-on 3G or pLVX-TRE-Flag-HNF4 alpha-Puro into HEK293T cells simultaneously according to a mass ratio of 3:1:4 by using a cationic polymer Polyethyleneimine (PEI) gene transfection technology, collecting a culture supernatant containing the lentivirus 72 hours after transfection, filtering the culture supernatant by using a 0.45 mu m filter membrane, concentrating the culture supernatant, and storing the culture supernatant at-80 ℃.
2) BEL-7404 cells are infected by the prepared hNTCP lentivirus, and are screened and identified by antibiotics.
3) The regulation was achieved by constructing a lentiviral infection system containing HNF 4. alpha. using the Tet-On system (Clontech, Mountain View, USA) and inducing expression of HNF 4. alpha. using this system via tetracycline (DOX). The BEL-7404-hNTCP cell screened in the last step is infected, and the purified BEL-7404-NTCP-Tet-on-HNF4 alpha (7404NT-HNF4 alpha) cell line is obtained after antibiotic screening.
2. Detecting transcript levels of hNTCP and HNF4 α in cells using a real-time quantitative pcr (qpcr) method:
1) RNA extraction was performed using a conventional Trizol extraction method.
2) RNA reverse transcription one-step cDNA was synthesized using the Tiangen FastKing method (Tiangen, KR 118).
3) Real-time quantitative PCR was performed using the Tiangen SYBR Green I chimeric fluorescence method (Tiangen, FP 205). Specific primers aiming at hNTCP and HNF4 alpha genes are adopted as amplification primers: hNTCP upstream primer: 5'-AAGGACAAGGTGCCCTATAAAGG-3' (SEQ ID NO: 1); a downstream primer: 5'-TTGAGGACGATCCCTATGGTG-3' (SEQ ID NO: 2); HNF4 α upstream primer: 5'-CGAAGGTCAAGCTATGAGGACA-3' (SEQ ID NO: 3); a downstream primer: 5'-ATCTGCGATGCTGGCAATCT-3' (SEQ ID NO: 4). The results are shown in FIG. 3A.
3. Western blot method for detecting protein expression levels of hNTCP and HNF4 alpha in cells
1) And (3) using SDS lysate to lyse cells, collecting cell lysate, centrifuging, taking supernatant, adding protein loading buffer, and denaturing at 100 ℃ for 5 min.
2) SDS-polyacrylamide gel electrophoresis, membrane transfer, blocking, antibody incubation [ anti- β -actin (Sigma-Aldrich), anti-HNF4 α (C11F12, Cell Signaling Technology, Beverly, USA) and anti-SLC10A1(Sigma-Aldrich) ] was performed, and after the corresponding secondary antibody incubation, visualization was performed with ECL Blotting Substrate (Millpore). The results are shown in FIGS. 3B-3C.
The result shows that the 7404NT-HNF4 alpha cell has higher hNTCP and HNF4 alpha expression level, and the expression of HNF4 alpha can be regulated and controlled by adding DOX in the culture medium.
Example 4
The expression and replication levels of HBsAg and HBeAg of HBV in 7404NT-HNF4 alpha cells are detected by enzyme-linked immunosorbent assay (ELISA) and Southern blot, and the infection level of HBV in 7404NT-HNF4 alpha cells is detected by ELISA:
1. after p1.3 XHBV replicon was transfected in 7404NT-HNF 4. alpha. cells, the expression levels of HBsAg and HBeAg in the supernatant were measured using ELISA test kit from Shanghai Kewawa, and the results are shown in FIG. 4A. Replication of HBV was detected using the method in example 2, and the results are shown in FIG. 4B.
2. 7404NT-HNF4 α cells were infected with concentrated HBV (MOI 1000) and 2.5% DMSO and 4% PEG8000 were added to the infection system. After 12h, the medium was changed, washed five times with PBS, and complete medium containing 2.5% DMSO was added, after which 48h the medium was changed and the supernatant was collected and assayed for HBeAg expression levels in the supernatant by ELISA. The results are shown in FIG. 4C.
The result shows that the expression levels of HBsAg and HBeAg of the 7404NT-HNF4 alpha cell are obviously improved under the induction of DOX, and the 7404NT-HNF4 alpha cell can support the replication of HBV and can be infected by the HBV.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.