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[Construction of plasmid stably expressing shRNA and inhibition of HBV replication and expression].

Hongbin Zhang, Jie Wu, Wenling Zheng, Wenli Ma, Jiang Xian, Wen-Yue Xie, Wei Li, Jie Wang

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Abstract

AIM: To construct the vector stably expressing small hair RNA(shRNA) and investigate the effect of shRNA expressed with DNA vector on HBV replication and expression. METHODS: The vector expressing shRNA designated pU6 was constructed by U6 promoter. The sequence of shRNA targeting on specific region of HBV genome against HBV was designed and synthesized, then cloned into pU6. The plasmid designated pU6B/HBVi was transfected into 2.2.15 cells using Lipofectamine 2000 reagent. The HBsAg and HBeAg in the supernatants of the transfected cells were measured by ELISA. The HBV copies in the transfected cells were measured by quantitative PCR. RESULTS: Compared with controls, not only concentration of HBsAg and HBeAg but also HBV copies in transfected 2.2.15 cells decreased significantly by the plasmid expressing shRNA against HBV. CONCLUSION: HBV replication and expression in 2.2.15 cells are inhibited by stably expressed shRNA.

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What this paper is about

AIM: To construct the vector stably expressing small hair RNA(shRNA) and investigate the effect of shRNA expressed with DNA vector on HBV replication and expression. METHODS: The vector expressing shRNA designated pU6 was constructed by U6 promoter. The sequence of shRNA targeting on specific region of HBV genome against HBV was designed and synthesized, then cloned into pU6. The plasmid designated pU6B/HBVi was transfected into 2.2.15 cells using Lipofectamine 2000 reagent. The HBsAg and HBeAg in the supernatants of the transfected cells were measured by ELISA. The HBV copies in the transfected cells were measured by quantitative PCR. RESULTS: Compared with controls, not only concentration of HBsAg and HBeAg but also HBV copies in transfected 2.2.15 cells decreased significantly by the plasmid expressing shRNA against HBV. CONCLUSION: HBV replication and expression in 2.2.15 cells are inhibited by stably expressed shRNA.

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Available abstract

AIM: To construct the vector stably expressing small hair RNA(shRNA) and investigate the effect of shRNA expressed with DNA vector on HBV replication and expression. METHODS: The vector expressing shRNA designated pU6 was constructed by U6 promoter. The sequence of shRNA targeting on specific region of HBV genome against HBV was designed and synthesized, then cloned into pU6. The plasmid designated pU6B/HBVi was transfected into 2.2.15 cells using Lipofectamine 2000 reagent. The HBsAg and HBeAg in the supernatants of the transfected cells were measured by ELISA. The HBV copies in the transfected cells were measured by quantitative PCR. RESULTS: Compared with controls, not only concentration of HBsAg and HBeAg but also HBV copies in transfected 2.2.15 cells decreased significantly by the plasmid expressing shRNA against HBV. CONCLUSION: HBV replication and expression in 2.2.15 cells are inhibited by stably expressed shRNA.

Key concepts: Small hairpin RNA, Lipofectamine, Transfection, HBsAg, HBeAg, Molecular biology, Plasmid, Virology

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