新基因XTP11剪切体的克隆与亚细胞定位

Cloning and subcellular location of the novel gene XTP11 spliced variant

[目的]克隆乙型肝炎病毒(hepatitis B virus,HBV)X蛋白反式调节基因11(XTP11)的剪切体,观察剪切体蛋白在人肝癌细胞HepG2中的定位。[方法]利用逆转录-聚合酶链式反应(reverse transciptase-polymerase chain re-action,RT-PCR)技术扩增XTP11剪切体,将目的基因片段插入克隆载体pGEM-T中,经双酶切和测序鉴定后,定向克隆至荧光表达载体pEGFP-C1中,转染HepG2细胞,荧光显微镜下观察确定其亚细胞定位。[结果]XTP1剪切体的开放阅读框长度为1314 bp,编码产物为437个氨基酸残基;重组质粒在HepG2细胞中转染效率为50%,蛋白定位于细胞浆。[结论]转染重组表达载体的细胞内出现局限性强绿色荧光信号,推断目的蛋白位于细胞质内。XTP11剪切体的克隆和亚细胞定位为进一步从分子水平分析其生物学功能奠定基础,为阐明其在乙型肝炎病毒X蛋白致病机制中的作用提供信息。

[ Objective] This study is to amplify the human gene 11 spliced variant transregulated by hepatitis B virus X antigen, and observe the subcellular location of new gene in HepG2. [ Methods ] RT - PCR was used to amplify XTP11 spliced variant, the products was cloned into pGEM -T vector. After restriction enzyme digestion analysis and sequencing, the correct DNA fragment was inserted into plasmid pEGFP - C1 （enhanced green fluorescent protein）. The recombinant plasmid was transfected to HepG2 cells and observed by inverted fluorescence microscope. [ Results] The new open reading frame（ORF） of XTP11 spliced variant is 1314 bp in length and translated a protein containing 437 amino acid residues;the efficiency of recombined plasmid transfection was 50% in HepG2, the location of protein was detected in cytoplasm. [ Conclusion ] XTP11 spliced variant can be subcelhdarly located in cytoplasm by its visible green fluorescent signal. These results pave the way for study the biological funcition of XTP11 spliced variant, providing information for clarifying the role of XPT11 spliced variant in the occurrence of HVB.