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针对HBVx基因的siRNA重组腺病毒的制备及其对HBVx基因表达的抑制作用
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作者：秦炜炜，刘家云，张瑞，王涛，王凯，孟艳玲，杨安钢
【关键词】 乙型肝炎病毒；,RNA干扰；,腺病毒；,基因治疗
Preparation of recombinant adenovirus mediated siRNA targeting HBVx gene and its suppressive effect on HBVx gene expression
【Abstract】 AIM: To construct the recombinant adenovirus vectors for siRNA targeting HBVx gene （HBx） and to evaluate the inhibitive effect on the expression of HBx gene in human hepatoma HepG2 cell line. METHODS: The shuttle vector pShuttleHBx was cotransfected into E. coli BJ5183 with adenovirus backbone plasmid. The homologous recombinants were transfected into HEK293 packaging cells and adenovirus was packaged and amplified, followed by infection of the HepG2 cells transfected with HBx gene. The expression levels of HBx were determined by RTPCR and Western Blot after the total RNA and protein were collected. RESULTS: The recombinant adenoviral vectors containing siRNA targeting HBx gene were successfully constructed, which were confirmed by restriction enzyme digestion. RTPCR results demonstrated that the HBx mRNA was markedly decreased and Western Blot results showed that the expression of HBx protein was also significantly reduced in HepG2 cell line. CONCLUSION: Adenovirusbased siRNA targeting HBx gene can inhibit HBx expression with great potency and specificity in vitro.
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