CN110669735A - System for inducing formation of HBV cccDNA and construction method - Google Patents

Abstract

The invention discloses a system and a construction method for inducing and forming HBV cccDNA, wherein a stable cell line is constructed by integrating Phic31 recombinase genes, and the 5 'end and the 3' end of linear HBV are provided with complementary luciferase gene fragments and specific sites recognized by Phic31 recombinase, and are cyclized to form cccDNA under the action of Phic31 recombinase, and simultaneously, the complementary luciferase is expressed along with the cccDNA; luciferase provides a shortcut for rapid detection of active HBV cccDNA, and is suitable for high-throughput screening.

Description

System for inducing formation of HBV cccDNA and construction method

Technical Field

The invention relates to a system for inducing and forming HBV cccDNA and a construction method, in particular to HBV cccDNA containing luciferase can be generated in liver cancer cells expressing PhiC31 recombinase by a linear genome, and the luciferase provides a shortcut for quickly detecting active HBV cccDNA and is suitable for high-throughput screening.

Background

Hepatitis B Virus (HBV) is a DNA virus belonging to the Hepadnaviridae (Hepadnaviridae) family. The HBV genome is about 3.2kb in total length and encodes 7 functional proteins: HBV core antigen (HBcAg), e antigen (HBeAg), surface antigen (L, M, S HBsAg), polymerase (pol) and X protein (HBx). There are now about 2.4 million people worldwide infected with HBV, of which there are perhaps 2500 million chronic infectors (Global Hepatitis Report,2017), which greatly increase the risk of cirrhosis and liver cancer, with over 70 million people dying from HBV-related diseases each year, which causes Hepatitis b to be one of the most serious public health problems worldwide.

After HBV enters hepatocytes through hepatocyte surface receptors, its viral nucleic acid (RC-DNA) is released into the cytoplasm, and immediately thereafter, the RC-DNA enters the nucleus to form covalently closed circular DNA (cccdna) or is directly integrated into the host genome. Among them, HBV cccDNA usually exists in the form of minichromosome, and can be used as a template to translate viral mRNA, including four 3.5kb mRNA (encoding core antigen, e antigen and polymerase), 2.4kb and 2.1kb S mRNA (encoding L, M, S three surface antigens), 0.7kb HBx mRNA. Wherein, 3.5kb pregenomic RNA (pgRNA) generates RC-DNA through reverse transcription, and finally forms complete virus particles to be released outside cells to complete the whole life cycle (Chen et al, 2015; Seegand Mason, 2015).

Clinical treatment of HBV at present stage usually involves two regimens of nucleoside analogues(s) analogues (NAs) and Interferon (IFN). Among them, nucleoside analogs can effectively inhibit the generation of new viruses by inhibiting reverse transcription links, and can not achieve the clinical effect of curing HBV by eliminating cccDNA in mechanism, and the risk of drug resistance is also existed in long-term administration, thereby reducing the curative effect (Zoulim and Locanini, 2009, 2012). Interferons inhibit viral replication and clear viruses mainly by activating the immune system, but the cure rate is very low (< 10%), far from meeting clinical needs; furthermore, interferon therapy is costly and has many non-adaptive limitations. Therefore, there is an urgent need to develop new therapeutic strategies with the aim of eliminating HBV cccDNA.

Currently, the cell models for HBV virus research are divided into two broad categories, infected and non-infected cell lines, which are mainly achieved by introducing non-viral vectors into the target cell line. In 1987, scientists in the United states used human hepatoma cell HepG2 to introduce pdolTHBV-1 plasmid containing double-length HBV genome and constructed the currently most commonly used non-infectious cell line HepG2.2.15(Sells et al, 1987), in which HBV gene was continuously expressed and HBV virus production was uncontrolled. In 2006 and 2007, the hepatitis virus institute, de lesel biotechnology and virology institute in the U.S. constructed two cell lines, HepAD38(Zhou et al, 2006) and HepDES19(Guo et al, 2007), which could induce the production of HBV, in which transcription of pgRNA of HBV is under the control of Tetracycline (Tetracycline); in the presence of tetracycline, pgRNA transcription is stopped, cells stop producing RC-DNA and HBV viral particles, and HBV cccDNA with relatively increased abundance can be obtained; the generation of these two cell lines greatly facilitated the understanding of HBV cccDNA. However, the efficiency of cccDNA formation by this method is generally low, and the real reaction of cccDNA in vivo cannot be simulated well.

In infected cell lines, cccDNA production can be efficiently induced by introducing HBV into a host cell line via a series of viral vectors or wild-type viral strains to induce cccDNA formation. In 2003, German scientists constructed HBV primary human liver cell lines using adenovirus vectors (Schulze-Bergkamen et al, 2003). Recently, the national institute of health selects human pluripotent stem cell differentiated hepatocyte-like cells to construct HBV infected cell lines (Xia et al, 2017), which enhance the research depth of cccDNA to some extent. However, since this cell line can efficiently produce cccDNA, which results in the formation of a large amount of useless cccDNA, while in hepatocytes of patients with hepatitis b, the copy number of cccDNA is low (average <1 copy/cell), and the excessive amount of cccDNA makes the immune response generated by host cells to be more different than that of hepatocytes of patients, and thus the physiological state of cccDNA cannot be truly reflected. In addition, the introduction of viral vectors can cause partial HBV genome integration into the genome of host cells, which can affect the judgment of experimental data in the later detection stage due to the inability to distinguish between genomic HBV and cccDNA, thereby leading to different conclusions.

In summary, direct research models of HBV cccDNA are rare, mainly due to: cccDNA and RC-DNA are highly similar on DNA sequence, and the copy number of HBV cccDNA in infected cells is far lower than that of RC-DNA, PCR detection cannot effectively distinguish HBV cccDNA, the Southern Blot detection mode has long time and low sensitivity; 2. the cell model and the animal model supporting HBV infection are lacked, the HBV cell infection model has lower efficiency and rigorous infection conditions, and the method is not suitable for high-throughput screening; the liver humanized mouse model is the only animal model supporting HBV infection, but has high cost and is only suitable for preclinical evaluation of drugs. Therefore, a simple and high-throughput technical platform for directly researching HBV cccDNA is urgently needed in the research and development field of HBV curative drugs.

Reference to the literature

1.Global Hepatitis Report 2017.World Health Organization,GenevaLicence: CC BY-NC-SA 3.0IGO

2.Chen,J.,et al.,2015.New insights into hepatitis B virus biology andimplications for novel antiviral strategies.Natl.Sci.Rev.2,296–313.

3.Seeger,C.,Mason,W.S.,2015.Molecular biology of hepatitis B virusinfection. Virology 479–480,672–686.

4.Zoulim F,Locarnini S.Hepatitis B virus resistance to nucleos(t)ideanalogues. Gastroenterology.2009；137:1593–1608.e1591–e1592.

5.Zoulim F,Locarnini S.Management of treatment failure in chronichepatitis B.Journal of hepatology.2012；56(Suppl 1):S112–S122.

6.Sells,M.A.,Chen,M.L.,Acs,G.,1987.Production of hepatitis B virusparticles in HepG2cells transfected with cloned hepatitis B virus DNA.Proc.Natl.Acad.Sci.U.S.A.84,1005–1009.

7.Zhou,T.,et al.,2006.Hepatitis B virus e antigen production isdependent upon covalently closed circular(ccc)DNA in HepAD38cell cultures andmay serve as a cccDNA surrogate in antiviral screening assays.Antivir.Res.72,116–124.

8.Guo,H.,et al.,2007.Characterization of the intracellulardeproteinized relaxed circular DNA of hepatitis B virus:an intermediate ofcovalently closed circular DNA formation.J.Virol.81,12472–12484.

9.Schulze-Bergkamen,H.,et al.,2003.Primary human hepatocytes–avaluable tool for investigation of apoptosis and hepatitis B virusinfection.J.Hepatol.38, 736–744

10.Xia,Y.,et al.,2017.Human stem cell-derived hepatocytes as a modelfor hepatitis B virus infection,spreading and virus-hostinteractions.J.Hepatol.66, 494–503.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a system and a construction method for inducing the formation of HBV cccDNA; the detection of HBV cccDNA is facilitated by introducing a luciferase reporter gene in HBV cccDNA.

In order to achieve the purpose, the invention adopts the technical scheme that: a system for inducing the formation of HBV cccDNA, comprising a liver cancer cell capable of expressing Phic31 recombinase, wherein the liver cancer cell comprises a linear genome formed by transfection, the linear genome comprises an HBV gene, the 5 'end and the 3' end of the HBV gene are respectively connected with a luciferase gene fragment and a Phic31 recombinase specific recognition site, the luciferase gene fragments connected with the 5 'end and the 3' end of the HBV gene constitute a complete luciferase gene, and the 5 'end and the 3' end of the HBV gene are respectively located at the 5 'end and the 3' end of the linear genome;

when the Phoc 31 recombinase expressed by the liver cancer cells induces the linear genome to form cccDNA, the cccDNA can be transcribed to form a complete open reading frame of a luciferase gene, and the luciferase is translated and expressed by the liver cancer cells.

As the improvement of the technical proposal, the liver cancer cell is HepG2 cell, and the nucleotide sequence of the HBV gene is shown as SEQ ID NO: 1; HBV gene is C genotype of China epidemic strain.

As an improvement of the technical scheme, the nucleotide sequence of the Phic31 recombinase is shown as SEQ ID NO. 2.

As an improvement of the technical proposal, the sequence of the luciferase gene fragment connected with the 5 'end of the HBV gene is shown as SEQ ID NO. 3, and the sequence of the luciferase gene fragment connected with the 3' end of the HBV gene is shown as SEQ ID NO. 4; the specific recognition sites of the Phic31 recombinase are attP and attB.

As a further improvement of the technical proposal, the Phic31 recombinase induces specific sites attP, attR sites formed by attB recombination and luciferase gene fragments Glu-N and Glu-C are positioned between the positions 85 and 86 after the initiation codon of the core antigen, and Glu-N and Glu-C are positioned at the outer sides; the sequence of the luciferase gene fragment Glu-N is shown as SEQ ID NO. 3, and the sequence of the luciferase gene fragment Glu-C is shown as SEQ ID NO. 4; the linear genome is transfected by ligation on an adenovirus plasmid.

In addition, the invention also provides HBV cccDNA formed by the system.

In addition, the present invention also provides a method for constructing a system for inducing the formation of HBV cccDNA, comprising the steps of: constructing liver cancer cells expressing Phic31 recombinase; constructing recombinant adenovirus plasmid connected with linear genome, packaging, purifying and transfecting liver cancer cell.

As an improvement of the technical scheme, the liver cancer cell expressing the Phic31 recombinase is constructed by the following method:

s11) replacing the Phyc 31 recombinase gene with the fluorescent protein gene EGFP by taking the plasmid pRlenti-Puro-EGFP as a vector to obtain a lentiviral vector plasmid pRenti-Puro-Phyc 31; the plasmid pRlenti-Puro-EGFP is a lentiviral vector with a screening gene Puro and a fluorescent protein gene EGFP, and the Puro-EGFP is connected by a self-shearing polypeptide 2A; the nucleotide sequence of the Phic31 recombinase is shown as SEQ ID NO. 2;

s12) lentivirus vector plasmid pRenti-Puro-Phic31, then the lentivirus pRenti-Puro-Phic31 is collected, the lentivirus pRenti-Puro-Phic31 is transfected into HepG2 cells, and HePG2-Phic31 cells which stably express Phic31 recombinase are screened.

As an improvement of the technical scheme, the recombinant adenovirus plasmid is constructed by the following method:

s21) connecting the luciferase fragment Glu-N and the luciferase fragment Glu-C to the 5 'end and the 3' end of the linear HBV gene respectively by adopting an overlap extension PCR method, thus constructing a Glu-N-HBV-Glu-C fusion fragment; the sequence of the luciferase fragment Glu-N is shown as SEQ ID NO. 3, and the sequence of the luciferase fragment Glu-C is shown as SEQ ID NO. 4;

s22) cutting an exogenous gene P2B9 of the PMC-P2B9 plasmid to obtain the PMC plasmid; the PMC-P2B9 plasmid is a T cloning vector with multiple cloning sites, and attP and attB recognition sites are respectively positioned at two ends of the multiple cloning sites; cloning the Glu-N-HBV-Glu-C fusion fragment onto a PMC plasmid, and carrying out transfection and purification to obtain a recombinant plasmid PMC-HBV-C-Gluc;

s23) taking the PGA1-empty plasmid as a skeleton vector, taking the PMC-HBV-C-Gluc as a target fragment, and cloning the HBV-C-Gluc fusion fragment to the PGA1-empty plasmid through enzyme digestion and connection to obtain a shuttle plasmid PGA 1-HBV-C-Gluc; the PGA1-empty plasmid does not carry exogenous genes;

s24) carrying out enzyme digestion on shuttle plasmid PGA1-HBV-C-Gluc and adenovirus plasmid AD5, recovering enzyme digestion products for transfection, and collecting recombinant adenovirus plasmid AD5-HBV-C-Gluc after transfection.

In addition, the invention also provides the system and/or the application of the HBV cccDNA in constructing an HBV drug screening system.

The invention has the beneficial effects that: the invention provides a system for inducing and forming HBV cccDNA and a construction method, wherein a stable cell line is constructed by integrating Phic31 recombinase genes, and the 5 'end and the 3' end of linear HBV are provided with complementary luciferase gene fragments and specific sites recognized by Phic31 recombinase, and are cyclized to form cccDNA under the action of Phic31 recombinase, and the complementary luciferase is expressed along with the cccDNA; the design of the invention has the following advantages: the liver cancer cells are preferably HepG2 cells, the HepG2 cells have simple biological background, the liver cancer cells are good vectors for researching hepatitis B virus and cccDNA, the quantity of introduced linear HBV can adjust the formation quantity of the cccDNA, the influence of a large quantity of useless cccDNA on an experiment is avoided, no virus is generated in the process, HBV can not be integrated into the genome of a host cell, the obtained experimental data can more intuitively reflect the state of the cccDNA, and expressed luciferase is secreted out of cells and can be detected by directly sucking a small amount of culture medium supernatant.

Drawings

FIG. 1 shows the transcript levels of the Phic31 recombinase gene in HePG2-Phic31 cells;

FIG. 2 shows the expression level of Phic31 recombinase in HePG2-Phic31 cells;

FIG. 3 shows the expression level of luciferase in HePG2-Phic31 cells;

fig. 4 is a schematic structural diagram of HBV cccDNA; wherein, the Phic31 recombinase induces specific sites attP, attR sites formed by attB recombination and two sections of luciferase gene fragments Glu-N and Glu-C are positioned between the positions 85 and 86 after the initiation codon of the Core gene, and Glu-N and Glu-C are positioned at the outer sides; when the Phic31 recombinase induces linear HBV to form cccDNA, Glu-N and Glu-C can be synthesized into Luciferase reporter gene Luciferase with complete ORF again, and the Luciferase reporter gene Luciferase is transcribed and translated to be expressed.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.

Obtaining genomic DNA from a patient sample

Hepatitis B virus RNA of a patient sample is extracted by using a nucleic acid extraction or purification kit (Daan gene at Zhongshan university), and the operation is as follows:

1) adding 200ul of sample into a centrifuge tube, and adding 50ul of protease K;

2) adding 200ul of cracking solution, performing vortex oscillation for 15sec, centrifuging for 10sec, and performing centrifugation at 72 ℃ for 10 min;

3) adding 250ul of absolute ethyl alcohol, and carrying out vortex oscillation for 15 sec;

4) sucking the mixed solution into a centrifugal column, centrifuging for 1min at room temperature at 12,000g, and transferring the centrifugal column to a new collecting pipe;

5) adding 500ul inhibitor removing solution into centrifugal column, centrifuging at room temperature for 1min at 12,000g, transferring the centrifugal column to new collection tube;

6) adding 500ul deionized liquid into a centrifugal column, centrifuging at room temperature for 1min at 12,000g, and transferring the centrifugal column to a new collection tube;

7) repeating the step 6;

8) centrifuging the spin column collection tube at 14,000g for 3min at room temperature to remove residual ethanol;

9) taking out the centrifugal column, placing in a new 1.5ml centrifugal tube, and placing for 2min at 72 ℃;

10) 50ul of eluent preheated at 72 ℃ is carefully added just above the membrane of the centrifugal column, the tube cover is tightly covered, the mixture is kept stand for 1min at room temperature, and 14,000g is centrifuged for 1 min.

Secondly, obtaining the C-type HBV fragment

The C-type HBV gene is amplified from the extracted and purified cDNA by utilizing the PCR technology, and the nucleotide sequence is shown as SEQ ID NO. 1.

By using

The Taq 2x Mix kit (NEB, M0287S) amplifies HBV type C, and the reaction solution is prepared according to the following ratio:

and reacted according to the following procedure: 1) denaturation 94 ℃ for 30 seconds, 2) annealing 60 ℃ for 30 seconds, 3) extension 68 ℃ for 3 minutes, 4) cycle number 35 times.

The C-type HBV gene was extracted from the PCR product using agarose gel recovery kit (Tiangen Biochemical technology). Wherein, the PCR product is separated according to the molecular weight by agarose gel electrophoresis, a single target DNA band is cut off from the agarose gel, and the cut single target DNA band is put into a clean centrifugal tube and weighed. To the gel block was added an equal volume of solution PN (100. mu.l of PN solution if the gel weighed 0.1g and the volume was considered to be 100. mu.l), and placed in a water bath at 60 ℃ with the centrifuge tube gently turned upside down to ensure adequate dissolution of the gel block. Adding the solution obtained in the previous step into an adsorption column CA2 (column equilibrium step: adding 500. mu.l of equilibrium solution BL into adsorption column CA2, centrifuging at 12,000rpm for 1min, pouring off the waste liquid in the collection tube, placing the adsorption column into the collection tube), standing at room temperature for 2min, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring off the waste liquid in the collection tube, and placing adsorption column CA2 into the collection tube. Add 600. mu.l of rinsing solution PW (check whether absolute ethanol has been added before use) to adsorption column CA2, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, dump the waste liquid from the collection tube, and place adsorption column CA2 into the collection tube. Centrifuge at 12,000rpm (13,400 Xg) for 2min to remove the rinse as much as possible. The adsorption column CA2 was left at room temperature for several minutes and thoroughly dried to prevent the residual rinse from affecting the next experiment. Placing the adsorption column CA2 in a clean centrifuge tube, suspending and dropwise adding appropriate amount of elution buffer EB into the middle position of the adsorption membrane, and standing at room temperature for 2 min. The DNA solution was collected by centrifugation at 12,000rpm (. about.13,400 Xg) for 2 min.

Thirdly, obtaining the truncated two-part fragment of the Luciferase

The plasmid containing the Luciferase is used as a template, the PCR technology is utilized to amplify the Luciferase into two parts, marked as Luciferase gene segments Glu-N and Glu-C, and the nucleotide sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

PCR products are recovered by an agarose gel recovery kit (Tiangen Biotechnology), and the specific recovery steps are detailed in step 2.

Fourthly, obtaining the fused HBV-Glu fragment

By utilizing the overlap extension PCR technology, Luciferase gene fragments Glu-N and Glu-C are respectively connected to the 5 'end and the 3' end of the HBV gene to construct Glu-N-HBV-Glu-C fusion fragments, and when HBV is cyclized, Glu-N and Glu-C form a complete Luciferase gene, thereby being used as a report system to prompt the formation of cccDNA. The PCR reaction conditions were as follows:

1)PCR Round1：

pre-denaturation at 94 deg.C for 5 min; denaturation, 30s at 94 ℃; annealing at 60 ℃ for 30 s; extension, 3min at 65 ℃ 20 s; 20 cycles; extending, 5min at 65 ℃; keep at 4 ℃ keep.

2)PCR Round2：

Pre-denaturation at 94 deg.C for 5 min; denaturation, 30s at 94 ℃; annealing at 60 ℃ for 30 s; extension, 3min at 65 ℃ 20 s; 35 cycles; extending, 5min at 65 ℃; keep at 4 ℃ keep.

Fifthly, obtaining the recombinant plasmid PMC-HBV-C-Gluc

The plasmid PMC-P2B9 is a T cloning vector (TaKaRa) with Multiple Cloning Sites (MCS), two elements attP and attB specifically recognized by a Phic31 recombinase are respectively positioned at two ends of the multiple cloning sites, the vector is used as a basic skeleton for transformation, and a self-carried exogenous gene P2B9 is excised, so that the PMC plasmid is obtained; the Glu-N-HBV-Glu-C fusion fragment was cloned on PMC plasmid. PMC-P2B9 is taken as a framework vector, Glu-N-HBV-Glu-C is taken as a fragment, the fragments are respectively cut by enzyme and then connected, and finally, a connection product is taken to transform competent cells (Tiangen biochemical technology), and the transformation process is as follows:

add 100. mu.l of competent cell suspension to a 1.5ml centrifuge tube and place on ice. Add 10ul ligation product, mix gently with a pipette, and let stand on ice for 30 min. And thermally shocking the mixture in a water bath at 42 ℃ for 90sec, and then rapidly putting the mixture on ice for 3-5 min. Adding 1ml LB liquid culture medium (containing 10g tryptone per liter yeast extract 5g, NaCl 5g, pH 7.0, no antibiotics, autoclaving), mixing, shaking culturing at 37 deg.C (180rpm) for 1 hr to restore the normal growth state of bacteria, and expressing plasmid-encoded antibiotic resistance gene. 100 mul of the bacterial liquid is taken and put on LB solid culture medium containing antibiotic Amp (15 g/L of agar for bacterial culture is added into LB liquid culture medium, the bacterial liquid culture medium is autoclaved, slightly cooled and then is led into a bacterial culture plate for continuous cooling), and the bacterial liquid is evenly coated. After the bacterial liquid is absorbed by the culture medium, carrying out inverted culture at 37 ℃ for 12-16 hours.

Sixthly, obtaining shuttle plasmid PGA1-HBV-C-Gluc and adenovirus plasmid AD5-HBV-C-Gluc

PGA1-empty is the shuttle plasmid necessary for constructing the adenovirus vector, does not carry any exogenous gene, takes the plasmid as the skeleton vector, PMC-HBV-C-Gluc is the target segment, utilizes the restriction enzyme of TaKaRa company to carry out enzyme digestion, and the reaction system is as follows:

reagent	Volume of
		Plasmids	10ug
10X M Buffer	2ul
		KpnI	1ul
NotI	1ul
		RNase-Free ddH2O	Make up to 20ul

PGA1 and HBV-C-Gluc were connected by using a Solutioni ligation kit from TaKaRa, the reaction time was 8 hours, and the system was as follows:

after the reaction is completed, TOP10 competent cells (Tiangen Biochemical company) are transformed, and the specific steps are shown in step five; colony PCR and enzyme digestion identify the correct clone.

Restriction enzymes SgrAI and SgrAI of TaKaRa, BstZ17I are used for enzyme digestion of PGA1-HBV-C-Gluc and adenovirus plasmid AD5-E1E3 respectively, the reaction time is 12 hours, after enzyme digestion products are recovered, BJ5183 competent cell transformation is carried out, and the two are recombined. The competence transformation step is shown in step five.

The transformed clone is subjected to agarose electrophoresis to greatly identify a recombinant plasmid (AD5 is 32K bp), and the successfully recombined clone is inevitably the large plasmid. And (3) identifying successful clone, then carrying out XL-Blue competent transformation, and limiting the growth of small plasmid by XL-Blue competent cells to achieve the effect of purifying large plasmid.

Clones after XL-Blue transformation were identified by using the restriction enzyme HindIII of TaKaRa, and agarose electrophoresis revealed the insertion of the target band.

Seventhly, packaging and purifying adenovirus AD5-HBV-C-Gluc

293T cells were plated in 15cm dishes and transfected at a cell density of 70% by replacing fresh medium (Gibco DMEM + 10% FBS) before transfection. The transfection reagent was Lipo3000, AD5-HBV-C-Gluc and Lipo3000 were diluted with Opti-MEM, mixed well and then left to stand at room temperature for 15 minutes, and then added gently to the cell dish medium without changing the medium. And after 48 hours, observing the pathological state of the cells, wherein cytoplasm is bright, cell nucleus is reduced, collecting the cells, repeating the steps in a liquid nitrogen-37 ℃ water bath for three times, and centrifuging to collect supernatant, namely virus supernatant. The virus supernatant was purified by cesium chloride gradient centrifugation and stored in a freezer at-80 ℃.

Eighthly, obtaining recombinase Phic31CDS fragment

The plasmid PT-Phic31 is a T cloning vector (Promega) with Phic31 recombinase gene, Phic31CDS is amplified by PCR technology by taking the plasmid as a template, the nucleotide sequence is shown as SEQ ID NO:2, and a reaction system and a kit are shown in a step two.

Ninthly, obtaining a lentiviral vector plasmid pRenti-Puro-Phic31

The plasmid pRlenti-Puro-EGFP is a lentiviral vector with a screening gene Puro and a fluorescent protein gene EGFP, and is constructed and modified by the laboratory, wherein the Puro-EGFP is formed by connecting self-cleavage polypeptide 2A (self-cleavage 2A peptide,2A), and the plasmid pRenti-Puro-Phic31 can be obtained by replacing a Phic31 recombinase gene with the fluorescent protein gene EGFP on the basis of the vector. And (3) performing SpeI and EcoRI (TaKaRa) double enzyme digestion on the Phic31PCR product and pRlenti-Puro-EGFP, fully reacting, and performing gel electrophoresis to recover enzyme digestion fragments, wherein the specific operation is shown in the step two. Ligation was performed using a Solutioni kit (TaKaRa), and TOP10 competent cell transformation was performed after 6 hours of reaction, as detailed in step five. And selecting the colony with good growth for PCR and enzyme digestion identification.

Ten, packaging and collecting of lentivirus pRenti-Puro-Phic31

293T cells were plated in 10cm dishes and transfected at a cell density of 70% by replacing fresh medium (Gibco DMEM + 10% FBS) before transfection. The transfection reagent was Lipo3000, and pRenti-Puro-Phic31 and Lipo3000 were diluted with Opti-MEM, mixed well and then allowed to stand at room temperature for 15 minutes, and then added gently to the cell dish medium without changing the medium. And collecting cell supernatant after 48 hours, namely virus supernatant.

Eleven, establishing a HePG2-Phic31 stable cell line

HepG2 cells were plated in 6cm dishes and infected at 90% cell density, with fresh medium (Gibco DMEM + 10% FBS) being replaced before infection. 2ml of the supernatant of pRenti-Puro-Phic31 virus was added to each 6cm dish, the medium was replaced after 12 hours with fresh medium and puromycin (Puro, 5mg/ml) was added at 48 hours with continued application of screening pressure until the control cells were completely apoptotic.

Identification of twelve, HePG2-Phic31 Stable cell lines

Real-Time PCR detection of Phic31RNA expression

HePG2-Phic31 and control HePG2 cells were collected and total RNA was extracted using TRIZOL lysis method. 1ml of TRIZOL solution was added to each sample, and after sufficient lysis, 0.2ml of chloroform was added thereto, followed by vigorous shaking for 30 seconds and standing at room temperature for 5 minutes. The supernatant liquid was aspirated, transferred to a new centrifuge tube, added to an equal volume of isopropanol, placed on ice for 10 minutes, and centrifuged at 12000g for 10 minutes. The supernatant was discarded, 1ml of 75% ethanol was added, the mixture was inverted upside down until RNA pellet was suspended, and then RNA was dissolved in 50ul of RNase-FreedH 2O, and if necessary, dissolved in water at 55 ℃ to 60 ℃ for 10 minutes. Using ReverTra

The kit (TOYOBO, FSK-101) carries out reverse transcription to synthesize cDNA, and the reaction system is as follows:

reagent	Volume of
		5x RT buffer	4ul
dNTP mixture(10mM)	2ul
		RNase inhibitor(10U/μl)	1ul
Oligo(dT)20	2ul
		RNA	2ug(～ul)
RNase-Free ddH2O	Make up to 20ul

The reaction procedure was as follows: the incorporated at 42 ℃ for 20min, the Heat at 99 ℃ for 5min, the stored activated solution at 4 ℃ or-20 ℃ using Ssoadvanced^TMUniversal

The GreenSupermix kit (BIO-RAD, 172-5274) detects the expression level of Phic31RNA of HepG2-Phic31 cell line.

As shown in FIG. 1, the invention succeeded in HePG2-Phic31 cells.

Western-blot detection of expression level of Phic31 protein

HePG2-Phic31 and control HePG2 cells were collected and total protein was extracted using RIPA lysis method. Adding 1ml of RIPA lysate to each sample to lyse cells to release protein, centrifuging at 14000g for 30 minutes, transferring the supernatant to a new centrifuge tube, adding 5 SDS-PAGE protein loading buffer, and heating in a boiling water bath for 3-5 minutes to fully denature the protein. SDS-PAGE gel electrophoresis was used to separate the proteins sufficiently, after which membrane transfer was performed, proteins were transferred from the gel to nitrocellulose (NC membrane) and then blocked to exclude non-specific sites on the membrane from binding to primary antibodies, typically by incubation overnight with prepared milk, PBST rinsed three times, washing away residual milk followed by incubation of the primary antibody for 1 hour 30 minutes, and finally by incubation with horseradish peroxidase (HRP) -labelled secondary antibody for 2 hours.

Detection of the Phic31 protein was performed using the Immobilon Western kit (Millipore, WBKLS 0500).

As shown in FIG. 2, the present invention succeeded in HePG2-Phic31 cells.

Thirteen, induction identification of cccDNA formation

HepG2-Phic31 and HepG2 cells are planted in a six-hole plate culture dish, an experimental group of HepG2-Phic31 + AD5-HBV-C-Gluc is set, a control group of HepG2-Phic31 and HepG2+ AD5-HBV-C-Gluc and adenovirus AD5-HBV-C-Gluc are infected with fresh culture medium (Gibco DMEM + 10% FBS) after 2 hours, and the culture medium is collected after 48 hours to detect Luciferase.

Detecting the Luciferase by using a Renilla Luciferase Assay System kit (Promega, E2810), mixing a sample to be detected with the same volume of a detection reagent, fully reacting, and then detecting in a multi-hole micropore plate luminescence detector

The fluorescence values were read.

As shown in FIG. 3, the experimental group HepG2-Phic31 + AD5-HBV-C-Gluc can express high-content luciferase, and the invention can well construct a system for inducing the formation of HBV cccDNA and is used for detecting the content of the HBV cccDNA; HBV cccDNA is shown in figure 4.

Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> eighth national hospital in Guangzhou City

<120> System for inducing formation of HBV cccDNA and construction method

<130>2019.06.06

<160>4

<170>PatentIn version 3.3

<210>1

<211>3215

<212>DNA

<213>Hepatitis B virus

<400>1

ctccacaaca ttccaccaag ctctgctaga tcccagagtg aggggcctat attttcctgc 60

tggtggctcc agttccggaa cagtaaaccc tgttccgact actgcctcac ccatatcgtc 120

aatcttctcg aggactgggg accctgcacc gaacatggag aacacaacat caggattcct 180

aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240

acagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccacgtgtcc 300

tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360

tcctggctat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420

atgcctcatc ttcttgttgg ttcttctgga ctaccaaggt atgttgcccg tttgtcctct 480

acttccagga acatcaacca ccagcacggg gccatgcaag acctgcacga ttcctgctca 540

aggaacctct atgtttccct cttgttgctg tacaaaacct tcggacggaa actgcacttg 600

tattcccatc ccatcatcct gggctttcgc aagattccta tgggagtggg cctcagtccg 660

tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720

tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780

gagtcccttt ttacctctat taccaatttt cttttgtctt tgggtataca tttaaaccct 840

aataaaacca aacgttgggg ctactccctt aacttcatgg gatatgtaat tggaagttgg 900

ggtactttac cgcaggaaca tattgtacta aaactcaagc aatgttttcg aaaattacct 960

gtaaatagac ctattgattg gaaagtctgt caaagaattg tgggtctttt gggctttgct 1020

gcccctttta cacaatgtgg ctatcctgcc ttgatgcctt tatatgcatg tatacaatct 1080

aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca atatctgaac 1140

ctttaccccg ttgcccggca acggtcaggt ctctgccaag tgtttgctga cgcaaccccc 1200

actggatggg gcttggccat aggccatcgg cgcatgcgtg gaacctttgt ggctcctctg 1260

ccgatccata ctgcggaact cctagcagct tgtttcgctc gcagccggtc tggagcgaaa 1320

cttatcggca ccgacaactc tgttgtcctc tctcggaaat acacctcctt tccatggctg 1380

ctagggtgtg ctgccaactg gatcctgcgc gggacgtcct ttgtctacgt cccgtcggcg 1440

ctgaatcccg cggacgaccc gtctcggggc cgtttggggc tctaccgtcc ccttcttcat 1500

ctgccgttcc ggccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560

tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620

tgaacgccca ccaggtcttg cccaaggtct tacataagag gactcttgga ctctcagcaa 1680

tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttaaagac tgggaggagt 1740

tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct 1800

gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1860

tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccgtataa 1920

agaatttgga gcatctgtgg agttactctc ttttttgcct tctgacttct ttccttctat 1980

tcgagatctc ctcgacaccg cttctgctct gtatcgggag gccttagagt ctccggaaca 2040

ttgttcacct caccatacag cactcaggca agctattctg tgttggggtg agttgatgaa 2100

tctggccacc tgggtgggaa gtaatttgga agacccagca tccagggaat tagtagtcag 2160

ctatgtcaat gttaatatgg gcctaaaaat cagacaacta ttgtggtttc acatttcctg 2220

tcttactttt ggaagagaaa ctgttcttga gtatttggta tcttttggag tgtggattcg 2280

cactcctccc gcttacagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340

tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400

atctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt agtatccctt 2460

ggactcataa ggtgggaaac tttactgggc tttattcttc tactgtacct gtctttaatc 2520

ccgagtggca aactccctcc tttcctcaca ttcatttaca ggaggacatt attaatagat 2580

gtcaacaata tgtgggccct cttacagtta atgaaaaaag gagattaaaa ttaattatgc 2640

ctgctaggtt ctatcctaac cttaccaaat atttgccctt agacaaaggc attaaaccgt 2700

attatcctga acatgcagtt aatcattact tcaaaactag gcattattta catactctgt 2760

ggaaggctgg cattctatat aagagagaaa ctacacgcag cgcctcattt tgtgggtcac 2820

catattcttg ggaacaagag ctacagcatg ggaggttggt cttccaaacc tcgacaaggc 2880

atggggacga atctttctgt tcccaatcct ctgggattct ttcccgatca ccagttggac 2940

cctgcgttcg gagccaactc aaacaatcca gattgggact tcaaccccaa caaggatcac 3000

tggccagagg caaatcaggt aggagcggga gcattcgggc cagggttcac cccaccacac 3060

ggcggtcttt tggggtggag ccctcaggct cagggcatat tgacaacagt gccagcagca 3120

cctcctcctg cctccaccaa tcggcagtca ggaagacagc ctactcccat ctctccacct 3180

ctaagagaca gtcatcctca ggccatgcag tggaa 3215

<210>2

<211>1818

<212>DNA

<213> artificially synthesized sequence

<400>2

atggacacct atgctggcgc ctatgaccgc cagtccaggg agagggagaa ctcctctgct 60

gcctcccctg ccacccaacg cagcgccaat gaggacaagg ctgctgacct gcagagggag 120

gtggagaggg atggcggcag gttcaggttt gtgggccact tctctgaggc ccctggcacc 180

tctgcctttg gcacagctga gaggcctgag tttgagagga ttctgaatga gtgcagggct 240

ggcaggctga acatgatcat tgtctatgat gtctcccgct tctcccgcct gaaagtcatg 300

gatgccatcc ccattgtctc tgagctgctg gccctgggcg tgaccattgt ctccacccaa 360

gagggcgtct tcaggcaggg caatgtgatg gacctgatcc atctgatcat gaggctggat 420

gcctcccaca aggagtcctc cctgaagtct gccaagatcc tggacaccaa gaacctgcag 480

agggagctgg gcggctatgt gggcggcaag gccccatatg gctttgagct ggtctctgag 540

accaaggaga tcaccaggaa tggcaggatg gtgaatgtgg tgatcaacaa gctggcccac 600

tccaccaccc ccctgaccgg cccatttgag tttgagcctg atgtgatcag gtggtggtgg 660

agggagatca agacccacaa gcatctgcca ttcaagcctg gctcccaggc tgccatccat 720

cctggctcca tcaccggcct gtgcaagagg atggatgctg atgctgtgcc caccaggggc 780

gagaccattg gcaagaagac agcctcctct gcctgggacc ctgccacagt gatgaggatt 840

ctgagggacc ccaggattgc tggctttgct gctgaggtga tctacaagaa gaagcctgat 900

ggcaccccca ccaccaagat tgagggctac aggattcaga gggaccccat caccctgagg 960

cctgtggagc tggactgtgg ccccatcatt gagcctgccg agtggtatga gctgcaggcc 1020

tggctggatg gcaggggcag gggcaagggc ctgtccaggg gccaggccat cctgtctgcc 1080

atggacaagc tgtactgcga gtgtggcgct gtgatgacct ccaagagggg cgaggagtcc 1140

atcaaggact cctaccgctg ccgccgccgc aaggtggtgg acccatctgc ccctggccag 1200

catgagggca cctgcaatgt ctccatggct gccctggaca agtttgtggc tgagaggatc 1260

ttcaacaaga tcaggcatgc tgagggcgat gaggagaccc tggccctgct gtgggaggct 1320

gccaggaggt ttggcaagct gacagaggcc cctgagaagt ctggcgagag ggccaacctg 1380

gtggctgaga gggctgatgc cctgaatgcc ctggaggagc tgtatgagga cagggctgct 1440

ggcgcctatg atggccctgt gggcaggaag cacttcagga agcagcaggc tgccctgacc 1500

ctgaggcagc agggcgctga ggagaggctg gctgagctgg aggctgctga ggcccccaag 1560

ctgcccctgg accagtggtt ccctgaggat gctgatgctg accccaccgg ccccaagtcc 1620

tggtggggca gggcctctgt ggatgacaag agggtctttg tgggcctgtt tgtggacaag 1680

attgtggtga ccaagtccac cacaggcagg ggccagggca cccccattga gaagagggcc 1740

tccatcacct gggccaagcc ccccacagat gatgatgagg atgatgccca ggatggcaca 1800

gaggatgtgg ctgcctag 1818

<210>3

<211>134

<212>DNA

<213> artificially synthesized sequence

<400>3

gtgaatggcg tgaaggtgct gtttgccctg atctgcattg ctgtggctga ggccaagccc 60

acagagaaca atgaggactt caacattgtg gctgtggcct ccaactttgc caccaccgac 120

ctggatgccg acag 134

<210>4

<211>430

<212>DNA

<213> artificially synthesized sequence

<400>4

gggcaagctg cctggcaaga agctgcccct ggaggtgctg aaggagatgg aggccaatgc 60

caggaaggct ggctgcacca ggggctgcct gatctgcctg tcccacatca agtgcacccc 120

caagatgaag aagttcatcc ctggccggtg ccacacctat gagggcgaca aggagtctgc 180

ccagggcggc attggcgagg ccattgtgga catccctgag atccctggct tcaaggacct 240

ggagcccatg gagcagttca ttgcccaggt ggacctgtgt gtggactgca ccaccggctg 300

cctgaagggc ctggccaatg tgcagtgctc tgacctgctg aagaagtggc tgccccagag 360

atgtgccacc tttgcctcca aaatccaggg ccaggtggac aagatcaagg gcgctggcgg 420

cgatgacacc 430

Claims (10)

1. A system for inducing the formation of HBV cccDNA, which comprises a liver cancer cell capable of expressing Phoc 31 recombinase, wherein a linear genome formed by transfection is contained in the liver cancer cell, the linear genome comprises HBV genes, the 5 'end and the 3' end of the HBV genes are respectively connected with a luciferase gene fragment and a Phic31 recombinase specific recognition site, the luciferase gene fragments connected with the 5 'end and the 3' end of the HBV genes form a complete luciferase gene, and the luciferase gene fragments connected with the 5 'end and the 3' end of the HBV genes are respectively positioned at the 5 'end and the 3' end of the linear genome;

when the Phoc 31 recombinase expressed by the liver cancer cells induces the linear genome to form cccDNA, the cccDNA can be transcribed to form a complete open reading frame of a luciferase gene, and the luciferase is translated and expressed by the liver cancer cells.

2. The system of claim 1, wherein the liver cancer cell is HepG2 cell, and the nucleotide sequence of the HBV gene is shown in SEQ ID NO. 1.

3. The system of claim 1, wherein the nucleotide sequence of the Phic31 recombinase is as set forth in SEQ ID NO 2.

4. The system as claimed in claim 1, wherein the sequence of luciferase gene fragment linked to the 5 'end of HBV gene is shown in SEQ ID NO. 3, and the sequence of luciferase gene fragment linked to the 3' end of HBV gene is shown in SEQ ID NO. 4; the specific recognition sites of the Phic31 recombinase are attP and attB.

5. The system of claim 4, wherein the Phic31 recombinase induces the specific sites attP, attR sites formed by attB recombination, and luciferase gene fragments Glu-N and Glu-C, located between positions 85 and 86 after the initiation codon of the core antigen, with Glu-N and Glu-C being on the outside; the sequence of the luciferase gene fragment Glu-N is shown as SEQ ID NO. 3, and the sequence of the luciferase gene fragment Glu-C is shown as SEQ ID NO. 4; the linear genome is transfected by ligation on an adenovirus plasmid.

6. HBV cccDNA formed systematically according to any one of claims 1 to 5.

7. A method for constructing a system for inducing the formation of HBV cccDNA, comprising the following steps: constructing liver cancer cells expressing Phic31 recombinase; constructing recombinant adenovirus plasmid connected with linear genome, packaging, purifying and transfecting liver cancer cell.

8. The method of claim 7, wherein the liver cancer cell expressing Phic31 recombinase is constructed by:

s11) replacing the Phyc 31 recombinase gene with the fluorescent protein gene EGFP by taking the plasmid pRlenti-Puro-EGFP as a vector to obtain a lentiviral vector plasmid pRenti-Puro-Phyc 31; the plasmid pRlenti-Puro-EGFP is a lentiviral vector with a screening gene Puro and a fluorescent protein gene EGFP, and the Puro-EGFP is connected by a self-shearing polypeptide 2A; the nucleotide sequence of the Phic31 recombinase is shown as SEQ ID NO. 2;

s12) lentivirus vector plasmid pRenti-Puro-Phic31, then the lentivirus pRenti-Puro-Phic31 is collected, the lentivirus pRenti-Puro-Phic31 is transfected into HepG2 cells, and HePG2-Phic31 cells which stably express Phic31 recombinase are screened.

9. The method of claim 7, wherein the recombinant adenovirus plasmid is constructed by:

s21) connecting the luciferase fragment Glu-N and the luciferase fragment Glu-C to the 5 'end and the 3' end of the linear HBV gene respectively by adopting an overlap extension PCR method, thus constructing a Glu-N-HBV-Glu-C fusion fragment; the sequence of the luciferase fragment Glu-N is shown as SEQ ID NO. 3, and the sequence of the luciferase fragment Glu-C is shown as SEQ ID NO. 4;

s22) cutting an exogenous gene P2B9 of the PMC-P2B9 plasmid to obtain the PMC plasmid; the PMC-P2B9 plasmid is a T cloning vector with multiple cloning sites, and attP and attB recognition sites are respectively positioned at two ends of the multiple cloning sites; cloning the Glu-N-HBV-Glu-C fusion fragment onto a PMC plasmid, and carrying out transfection and purification to obtain a recombinant plasmid PMC-HBV-C-Gluc;

s23) taking the PGA1-empty plasmid as a skeleton vector, taking the PMC-HBV-C-Gluc as a target fragment, and cloning the HBV-C-Gluc fusion fragment to the PGA1-empty plasmid through enzyme digestion and connection to obtain a shuttle plasmid PGA 1-HBV-C-Gluc; the PGA1-empty plasmid does not carry exogenous genes;

s24) carrying out enzyme digestion on shuttle plasmid PGA1-HBV-C-Gluc and adenovirus plasmid AD5, recovering enzyme digestion products for transfection, and collecting recombinant adenovirus plasmid AD5-HBV-C-Gluc after transfection.

10. The system of any one of claims 1 to 5, and/or the use of HBV cccDNA of claim 6 in the construction of HBV drug screening system.

Images