Construction Of Several Special Cell Lines Of Hepatitis B Virus Mutantants With Inducible Expression

Hepatitis B virus (HBV) infections are a major worldwide health problem with Chronic infections leading to cirrhosis and hepatocellular carcinoma. Accompanying the widespreadly vaccination and generally application of antiviral drugs, the morbidity and mortality have been significantly reduced, but several problems arise such as vaccine /diagnostic escape mutantion and nucleotide analog resistant mutation, leading to examination and treatment failure. Compared to wild strain of HBV, there must be particular character in the aspect of bionomics of mutants. In order to deeply understand the affect to levels of virus replication and vaccine efficacy by these alterations, study the mechanisms of resistance to NA, and eventually develop novel antiviral agents, we need to construct several special cell models of HBV mutants. It is reported in domestic and abroad, two human hepatoma cell lines, HepG2 and Huh7, can support virus replication when transiently transfected with HBV DNA. However, it is not applicable for particular applications requiring standardized conditions such as drug screening because it lack of ability to replicate stably. As all known, HepG2.2.15 is a extensively applicatied HBV cell line, and widely used in antiviral research, providing distinct advantages: essentially all cells are productive, inter-experiment variation is minimized, and identical cells producing identical virus are relatively easily maintained and expanded. But HBV production is limited and difficult to control. Our aim is to establish stable, inducibly HBV mutantants producing cell lines that overcome these limitations. Tetracycline (Tet)-inducible expression system is one of the most prominent and widely-accepted inducible systems in eukaryotes so far. On this study, firstly, we constructed several full-length genome hepatitia B virus plasmids with Site-directed mutagenesis by PCR, enzyme-cutting recombination technique and other molecular biology skills. Secondly, tTA cells were transfected which were successedly constusted previously, and then by screening stable clones with hygromycin to construct several special cell lines of Hepatitis B virus mutants with inducible expression. Thirdly,we used two NAs to assess the suitability of the new cell lines by testing HBV inhibitors. It may be useful not only for diagnosis of Hepatitis B virus infection, but also as a valuable tool for HBsAg variant detection. In addition, it establish foundation for studying the mechanisms of resistance, and screening for new antiviral drugs. This study includes two parts.Part one Construction of several special cell lines with inducible expressionObjective: To construct 8 full-length genome hepatitia B virus plasmids with Site-directed mutagenesis by molecular biology technique, HBV vector was cloned into plasmid pTRE2hyg. Through transfecting tTA cells successedly constusted previously, we can also construct several special cell lines of HBV mutants with inducible expression. The ability of stable replication and regulation can be assessed by analysising the level of HBV expression.Method:1 To make Site-directed mutagenesis in the B, C, D domain of HBV pol (173, 180, 181, 204, 236) and HBsAg (100, 120, 145) by PCR and enzyme-cutting recombination technique. Through cloning this HBV vectors into plasmid pTRE2hyg, we can construct 8 plasmids with hygromycin-resistant, including pTRE-HBV-A181T, pTRE-HBV- N236T, a) pTRE-HBV-A181T-N236T, pTRE- HBV-M204I, pTRE-HBV-L180M- M204V, pTRE-HBV-V173L-L180M-M204V, pTRE-HBV-G145R, pTRE- HBV-Y100C-P120T.2 After identifying by enzyme-cutting and sequencing, the plasmids should be prepared largely.3 Previously, we have successedly constusted one new HepG2 cell line, named G2TA2-7, which was cloned with plasmid ptTA2. It was respectively transfected with this 8 plasmids newly construsted and plasmid pTRE-WMHBV. DOX was added and maintained until resistant clones became visible.4 Initial screening for HBV expression was done by testing HBV DNA and WMHBV DNA.5 After 20 generations without any antibiotics, the replication ability of individual clones were monitored by southern blot. Eventrolly, best cell lines should be selected.6 Regulatability was monitored by Southern blotting for nucleocapsid associated HBV DNA in cells grown without, or with doxycycline.7 To examine the influence to replication of HBV mutants by transfection technique and southern blot.Result:1 The eight mutants with hygromycin-resistant were successfully constructed.2 After transfection, about 60-100 stable transfectants each were selected using hygromycin.3 3 48 individual clones were picked from every 60 stable transfectants to be cultureed expandly. Eventually, 4 better clones were seclected by testing HBV DNA by PCR in the culture supernatants.4 4 After persistly cultured without any antibiotics for 20 generations(about 4months), replicative intermediates were monitored by Southern blot. In comparison to HepG2.2.15 cells, all new cell lines produced particularly stronger signals. Eventually, 9 best cell lines stood out as it yielded up to higher levels than many other clones, named A181T55, N236T13, A181T-N236T1, M204I43, L180M-M204V16, V173L-L180M-M204V44, G145R1, Y100C-P120T3 and WMHBV42.5 All Clones providing high level and tightly regulated expression were identified by southern blot. Without Dox, replicative intermediates can be seen, yet were still tightly suppressed by Dox addition.6 Compared to HBV wild strains,the replication ability of mutants of M204V, M204I and N236T is obviously decressing.However,it changed a little in mutants of L180M-M204V, G145R and Y100C-P120T.Conclusion: Successful construction of several special cell lines with inducible expression, is helpful for studying the function of any gene in different stages of development. It also establish foundation for screening for new antiviral drugs, and accurate diagnosis of Hepatitis B virus infection. Part two Assess the suitability of the new cell lines for testing HBV inhibitorsObjective: To test the sensibility of cell lines of HBV mutants and WMHBV cell line for LAM and ADF.It may benefit for optimizing methods of diagnosis, and selecting new antiviral drugs.Method:1 Assess the sensibility of the cell lines of nucleotide analog resistant newly construsted to LAM and ADF by southern blot.2 Examine the sensibility of the cell lines newly construsted to LAM and ADF by Native southern.3 Detect the sensibility of ADF theory resistant mutants to ADF by plasmid transient transfection and southern blot.4 To examine the correctness of the sites of Site-directed mutagenesis resistant to ADF by PCR and sequencing technique.Result:1 By Southern blot it can be easily seen that the signals of replicative I ntermediates in the group of A181T55, N236T13, A181T-N236T1 and HepG2.117 are gradually weakened,follwing by adding bigger drug concentration of LAM. However, the replication abilities of L180M-M204V16, M204I43and V173L-L180M-M204V44 are not affected. All cell lines including A181T55, N236T13 and A181T-N236T 1 are Sensitive to ADF.2 By Native Southern, we can find that wide type HBV, G145R1, Y100C-P120T3 and WMHBV 42 are sensitive to both LAM and ADF. The signals of L180M-M204V16 and M204I43 are not influenced by adding LAM. The abbilities of all cell lines, including N236T13 to replicate become lower after adding ADF.3 By plasmid transient transfection and southern blot, we can find N236T is sensitive to ADF,resemble to wide HBV.4 By sequencing, we can find Site-directed mutagenesis at site 181 in A181T55, site236 in N236T13, and site 181, 236 in A181T-N236T1, which is correspond to designing before.Conclusion: As cell model,we partly test the sensitivity to frequent HBV inhibitors. It may establish foundation for screening for new antiviral drugs, constructing animal model,and carrying put experiment invivo.