乙型肝炎病毒x基因蛋白转导载体的构建

乙型肝炎病毒X蛋白在细胞转化和肝癌的发生发展中具有重要作用.为了深入研究X蛋白的致癌机理构建了pTAT-GFP-X载体.该研究以克隆在真核表达载体pCMV-X质粒中的x基因为模板,设计了x基因的PCR引物,采用PCR方法扩增x基因,回收PCR产物,以XhoI和EcoRI酶切位点将PCR产物连接到蛋白转导系统pTAT-GFP载体中,再用XhoI和EcoRI酶对筛选的重组子进行酶切鉴定,获得了510bp的目的片段,表明已成功地将目的片段克隆在pTAT-GFP载体中.经DNA序列分析检测,显示克隆的x基因无突变.经SDS-PAGE和Westernblot检测证实,将重组质料pTAT-GFP-X转化致大肠杆菌BL21中,可表达pTAT-GFP-X融合蛋白.该pTAT-GFP-X载体蛋白转导系统的构建,为进行X蛋白的蛋白转导实验奠定了基础.pTAT-GFP-X融合蛋白具有穿透细胞膜进入细胞的能力,进而在细胞内发挥作用,与转基因不同,无需细胞内基因表达的过程,使得X蛋白的定量实验成为可能,X蛋白的蛋白转导实验更有助于探讨x基因的致癌机理.

Construction of a Recombinant Plasmid of pTAT-GFP-X Cloned Hepatitis B Virus x Gene, a Protein Transduction System

Hepatitis B virus (HBV) X protein plays a crucial role in the cellular transformation and occurrence of hepatocellular carcinoma. In order to illustrate the carcinogenesis of hepatoma by HBX protein, we constructed a recombinant plasmid of pTAT-GFP-X. The pCMV-X plasmid cloned full length x gene was used as templet for PCR. And the PCR primers of HBX gene were designed according to the full length sequence of x gene. The PCR product of x gene was amplified and reclaimed, which was connected to the protein transduction system, pTAT-GFP vector, using Xho I and EcoR I enzymatic sites. The same enzymes were used to identify the recombinant plasmid. The 510 bp target fragment obtained suggests that the target fragment of the x gene was successfull inserted into the pTAT-GFP vector. DNA sequencing analysis indicated that there were no any mutations in the cloned x gene. The recombinant plasmids of pTAT-GFP-X were transformed into the BL21 bacteria, followed the fusion protein of TAT-GFP-X contained HBX protein was identified by SDS-PAGE and Western blot analysis. The construction of the recombinant plasmid of pTAT-GFP-X was significant for the HBX protein transduction experiment. Fusion protein expressed by the plasmid in E coli is available to cross the cell membrane and to perform its function in the cells. Different from the gene transfection, the protein transduction doesn't acquire the gene expression in the cells. Therefore, it is possible to control the quantity for HBX protein in the cells, and the HBX protein transduction experiment is more helpful to understand the role of HBX protein in carcinogenesis of hepatoma.