摘要
目的:利用HIV-Tat蛋白转导域(proteintransductiondomain,PTD)高效的跨膜转导特性,探索将乙肝病毒靶向核糖核酸酶(targetedribonuclease,TR)导入肝细胞的新途径.方法:将TR基因克隆入含TatPTD的pTAT原核表达载体,在大肠杆菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.将纯化的Tat-TR加入培养的HepG2细胞,间接免疫荧光法检测其导入HepG2细胞的效率,MTT法检测其对细胞生长代谢的影响.将Tat-TR加入培养的HepG2.2.15细胞,定量PCR法检测HBVDNA水平并以SPSS软件进行统计分析.将Tat-TR经尾静脉注入小鼠体内2h后,对小鼠肝组织进行免疫组化染色,检测TatPTD能否在体内介导TR进入肝细胞.结果:成功地制备了Tat-TR融合蛋白,其纯度为90%.间接免疫荧光及MTT的检测结果证实Tat-TR可以跨膜导入HepG2细胞且对细胞生长没有影响.SPSS统计分析显示Tat-TR可以抑制HBV的复制.免疫组化染色结果显示,TatPTD在体内也可以介导TR进入肝细胞.结论:Tat-HBV靶向核糖核酸酶的制备及活性鉴定,为应用靶向核糖核酸酶治疗乙肝病毒感染的研究奠定了良好的基础.
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.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第8期958-962,共5页
World Chinese Journal of Digestology
基金
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~~
