人B7.2(CD86)胞外区原核表达及其工程菌发酵培养的实验研究  

Prokaryotic expression of extracellular domain of B7.2 (CD86) and fermentation study on the engineered bacteria

作　　者：闫晓彩[1] 来宝长[1] 郑瑾[1] 马军[1] 韩俊宏[1] 王一理[1] 司履生[1] 

机构地区：[1]西安交通大学医学院免疫病理研究室,西安710061

出　　处：《西安医科大学学报》2002年第1期4-7,共4页Journal of Xi'an Medical University(Chinese)

摘　　要：目的　利用原核系统表达人B7.2 (IgV +C)并对工程菌发酵培养条件进行优化。方法　用聚合酶链反应 (PCR)技术从B7.2cDNA全长中克隆B7.2 (IgV +C) ,将PCR产物克隆入表达载体 pGEX 4T 3从而得到重组体 pGEX 4T 3 /hB7.2 (IgV +C) ,SDS PAGE及Westernblot用于检测目的蛋白的表达 ,同时对目的蛋白诱导表达时间及诱导剂浓度进行优化 ,对重组质粒的遗传稳定性进行鉴定。结果　Westernblot结果显示相应分子质量 5 5kD处有hB7.2 (IgV+C)与GST融合蛋白的高效表达 ,表达量占菌体总蛋白的 3 0 %左右。对工程菌进行发酵培养研究的结果表明 ,所构建的重组质粒在工程菌DH5α中传代稳定 ,未见质粒丢失 ,目的蛋白的表达不受影响 ,且在 3 7℃诱导 5h、IPTG终浓度为 4mmol·L- 1 时其表达量最多。结论　证明了利用原核系统表达人B7.2 (IgV +C)的可行性。Objective To express the extracellular domain of human B7.2 (CD86), B7.2 (IgV+C),in prokaryotic expression system and to optimize the fermentation for engineered bacteria. Methods B7.2 (IgV+C) was amplified by PCR from B7.2 whole cDNA, and then was cloned into an expression vector pGEX 4T 3 to form a recombinant pGEX 4T 3/hB7.2 (IgV+C). SDS PAGE and Western blot were performed to identify the expression of the target protein. The optimal induction duration and the optimal inducer concentration were also selected and the inheritance stability of the recombinant plasmid was studied. Results After being transformed into E.coli DH5α, recombinant pGEX 4T 3/hB7.2 (IgV+C) was induced by IPTG to express a 55 Kda fusion protein in E.coli at a level of about 30% of the total cellular protein. The fermentation study showed that the recombinant plasmid was inherited steadily in DH5 α,with a stable expression yield of targeted protein and without loss of recombinant plasmid,and the expression yield peak could be seen at 5 hours after induction and 4?mmol·L -1 IPTG concentration. Conclusion These findings presented the possibility of expressing the human B7.2 (IgV+C) molecule in prokaryotic expression system.

关 键 词：B7.2(CD86) 共刺激 融合蛋白 GST 工程菌发酵培养 实验研究 抗原 原核表达 

分 类 号：R392.11[医药卫生—免疫学]