HDV核酶对乙型肝炎病毒抑制作用的研究

Investigation on the suppression of HBV by trans-acting HDV ribozyme in HepG2.2.15 cell

目的研究HDV核酶在细胞内对乙型肝炎病毒(HBV)复制及其抗原表达的抑制作用。方法1)以HBV前基因组mRNA为靶基因,体外筛选出HDV核酶有效作用位点,构建HDV核酶并进行体外测活;2)分别选用tRNA-Val、U6和hCMV 3种真核启动子,重组构建HDV核酶真核表达载体ptVHRz、pSURz和pcDHRz,分别用3种载体转染HepG2.2.15细胞;3)用点杂交、ELISA和实时荧光定量PCR方法分别检测核酶在细胞内的表达及对HBV的抑制作用。结果在HBV C基因区筛选到一位点,所构建的HDV核酶在体外条件下对该位点能产生有效切割。3种核酶表达载体在细胞内均能高效表达,在转染48 h后,ptVHRz和pcDHRz对HBeAg的表达产生了明显的抑制作用,而对HBsAg没有抑制作用。三者对HBV的复制均未产生明显影响。结论HDV核酶在细胞内对HBV抗原的表达能产生特异性抑制作用,但未能有效抑制HBV的复制,对其原因需进一步深入研究。

Objective To investigate the ability of trans-acting HDV （hepatitis delta virus） ribozyme to suppress the replication of hepatitis B virus （HBV） in vivo. Methods 1 ） Selection of potential HDV ribozyme target sites within the pregenome of HBV in vitro ： an HBV genome full sequence was cloned from HepG2. 2. 15 cell＇s genomic DNA, and aligned with 202 HBV full sequences of various genotypes which were retrieved from GenBank database, potential target sites which conserved in most of the genotypes （homology 〉 90 %） were identified. Several sites located in C region were examined by RNase H hydrolysis with oligonucleotides （18nt） and one site that may be accessible for the ribozyme was selected. Cleavage activity of the trans-acting HDV ribozyme constructed in our lab for this site was tested in vitro; 2） Construction of HDV riboxyme expression vectors： the cDNA of HDV ribozyme was recombined with three eukaryotic promoters, tRNA^Val, hMCV and U6, and three vectors ptVHRz, pSURz and pcDHRz were constructed respectively. Then they were transfected respectively into HepG2.2.15 cells by LipofectomineTM2000 （Invitrogene） ; 3） Assay： the expression of HDV ribozymes in cells was assayed by Northern blot dot hybridization; HBsAg, HBeAg and HBV in the cultural supernatant were assayed by ELISA and real time quantity PCR respectively. Results The selected site can be effectively cleaved by the constructed ribozyme in vitro. When transfected into HepG2.2.15 ceils after 48 hours, three vectors were all expressed highly and two of them, ptVHRz and pcDHRz, have significantly inhibited the expression of HBeAg but not for HBsAg. However no any suppression for the replication of HBV was observed in the study. Conclusion The ability of trans-acting HDV ribozyme specially suppressing the expression of HBeAg in vivo shows that it is a new potential tool for HBV gene therapy. But the fact that the replication of HBV was not suppressed by the HDV ribozyme is remained to elucidate and further work needs to be done.