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《World Chinese Journal of Digestology》 2005-08
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TatPTD can introduce HBV targeted ribonuclease into hepatocytes

Jin Ding, Jun Liu, Cai-Fang Xue, Ying-Hui Li, Ya Zhao, Jun Chen, Yu-Xiao Huang, Zhong-Xiang Liu, Department of Etiology, the Fourth Military Medical University, Xi'an 710032, Shaaxi Province, China  
AIM: To introduce hepatitis B virus(HBV)targeted ribonu-clease into hepatocytes by using human immunodeficiency virus Tat protein transduction domain (HIV-TatPTD). METHODS: The gene encoding HBV targeted ribonuclease was cloned into prokaryotic expression vector pTAT-HA. Recombinant plasmid was transduced into E.coli BL21 (DE3) LysS, and then induced with IPTG. The expression of the fusion protein was analyzed by SDS-PAGE. Tat-TR fusion protein was purified and added to the HepG2 cell culture. The transduction efficiency was evaluated by IFA. MTT assay was performed to investigate the cytotoxicity of Tat-TR. Tat-TR was added into the cultured HepG2.2.15 cells, and the HBV DNA concentration was measured using quantitative PCR. Immunohistochemical staining was performed to identify whether TatPTD could introduce TR into the hepatocytes in vivo. RESULTS: Tat-TR fusion protein was successfully expressed and purified. IFA visualization showed that Tat-TR was introduced into hepatocytes. MTT assay suggested that Tat-TR did not affect the cell growth and had no cytotoxity. Quantitative PCR result indicated that Tat-TR inhibited the replication of HBV. Immunohistochemical staining result showed that TatPTD delivered TR into hepatocytes in vivo. CONCLUSION: The Tat-TR fusion protein may be valuable for the therapy of HBV infection.
【Fund】: 国家自然科学基金资助课题 No.30100157 全军医药卫生科研基金资助课题 No.01MA184 第四军医大学创新工程资助课题 No.CX99005~~
【CateGory Index】: R373
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