Establishment Of The Hepatocellular Carcinoma Cell Line Stably Transfected With Hepatitis D Virus

Hepatitis D virus(HDV)is a viroid-like ribonucleic acid(RNA)virus,which requires hepatitis B virus(HBV)to provide hepatitis B virus surface antigen(HBsAg)for the assembly,secretion and infection of the virus.Hepatitis B chronic infection is a major cause of liver cirrhosis and hepatocelluar carcinoma,and HDV superinfection confers additional risk.But the current research on HDV is still relatively lacking,so more study of HDV is needed in future.At present,there are two ways to obtain hepatitis D virus: one way is to directly extract it from the sera of patients with hepatitis D.However,patients with hepatitis D in China are relatively rare,so if you want to rely on the direct purification of HDV from the serum of them,the result will be not very satisfactory.The other way is the HDV packaging method based on the plasmid transiently transfected HuH-7 and other cell lines.However,when the study requires a large number of viruses,the workload will increase significantly,and the reproducibility between experimental batches is also poor.Recently,Xiamen University published a new method for HDV virus packaging based on adenovirus,which solved a certain portion of the problem.But so far there is no HDV stable packaging cell line for HDV mass production and antiviral drug evaluation.HDV is a satellite virus of HBV,providing an alternative tool for HBV infection research.In this study,a piggyBac(PB)transposon vector containing HDV replicon and HBsAg expression cassette was constructed by molecular cloning method and the functional evaluation was carried out.The constructed vector was transiently transfected into HuH-7 cells.The function of the vector was determined by detecting the contents of HDV RNA and HBsAg in the cell supernatant.The related functional genes were expressed effectively.Supernatant was enriched and infected HepG2.N9 cell line with Na+/taurocholate Cotransporting Polypeptide(NTCP)-overexpressing.The cell supernatant was identified to contain infectious HDV particles.The research group members have transfected many human-derived hepatocarcinoma cell lines using this vector and other HDV replication packaging systems,and finally a high-efficiency cell line K7 has been obtained by rapid and large-scale screening with puromycin.In this study,immunofluorescence was used to detect the expression of HDV virus delta antigen and HBV surface antigen in K7 cells.The content of HBV surface antigen in supernatant was detected by ELISA.The titer of HDV virus was detected by quantitative PCR.The NTCP stably transfected cell line HepG2.N9 was used to evaluate the vitro infectivity of HDV virus secreted by the K7 cell line.The results showed that HDV virus delta antigen and HBs Ag expressed efficiently in the K7 cells,and the cell supernatant contains large amount of HDV particles with titer above 1E+08GE/ml(GE,genome equivalents),and the HDV virus secreted by this cell line have a good infection ability in vitro.In summary,we constructed an HDV expression vector containing HDV replicon and HBsAg expression frame and established a human hepatocellular carcinoma cell lines named K7 to support the replication,packaging and secretion of HDV with high efficiency in this study.This K7 cell line facilitated HDV large-scale production,and provided a platform for large-scale screening of potential anti-hepatitis D molecular drugs in future.