乙型肝炎病毒前S1蛋白反式调节基因2的克隆、表达及功能

Protein expression and function of gene 2 transregulated by hepatitis B virus pre-sl protein and its cloning

目的应用原核表达系统表达HBV前S1蛋白反式调节基因2（PS1TP2），酵母双杂交技术筛选白细胞文库中与之结合的蛋白质，了解该基因产物在肝癌细胞系中的亚细胞定位。方法应用生物信息学初步探讨PS1TP2结构及功能。PCR扩增PS1TP2基因，构建pET32a（＋）-PS1TP2表达载体，在大肠埃希菌Rosettap中进行诱导表达。构建pGBKT7-PS1TP2重组载体，应用酵母双杂交技术，在营养缺陷型培养基上筛选白细胞cDNA文库中与之结合的蛋白质。构建pEGFPC1-PS1TP2表达质粒，转染肝癌细胞株HepG2，利用荧光显微镜和共聚焦显微镜观察PS1TP2的亚细胞分布。结果PS1TP2基因位于6q24．1，疏水指数较高。PS1TP2能在原核系统中表达，表达产物相对分子质量约4．1×10＾4。酵母双杂交技术筛选出白细胞cDNA文库中与之结合的蛋白质26个。PS1TP2亚细胞定位于细胞质中。结论在原核表达系统中成功表达了PS1TP2，为抗体的制备奠定了基础。PS1TP2可与白细胞表达的某些蛋白质相互作用，在肝癌细胞系中定位于细胞质，为进一步探究HBV感染的发病机制提供了新线索。

Objectives To screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells. Methods The function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a （＋） and pGBKT7 to construct recombinant expression vectors pET32a （＋）-PS 1TP2 and pGBKT7-PS 1TP2. They were transduced into E. coli Rosetta strain and yeast AH 109. The transformed yeast mated with yeast Y 187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） for selecting twice and then screening. Then a green fluorescent protein （GFP） expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy. Results Bioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH 109, the expression protein showed： （1） the molecular weight of the expressed product was about 41 000 Da, and （2） PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1- PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma. Conclusion A prokaryotic expression vector pET32a（＋）-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.