构建Loa22基因去信号肽片段原核重组表达载体  

Constructing a prokaryotic expression vector of Loa22 gene excluding signal peptide

作　　者：朱海龙[1] 鲍朗[1] 张会东[1] 

机构地区：[1]四川大学华西医学中心感染免疫研究室

出　　处：《中国组织工程研究与临床康复》2007年第37期7358-7360,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30471546)~~

摘　　要：目的:构建赖型钩端螺旋体OmpA膜蛋白Loa22基因去信号肽片段的原核表达载体,并对其进行克隆表达。方法:实验于2004-12/2005-12在四川大学华西医学中心感染免疫研究室完成。以赖型钩端螺旋体017株基因组DNA为模板,PCR扩增Loa22基因去信号肽片段,亚克隆至原核表达载体pGEX-4T-1,经双酶切、PCR鉴定,筛选出阳性重组质粒克隆。经DNA测序正确后,转化大肠杆菌,利用IPTG进行诱导表达,通过SDS-PAGE鉴定表达产物。结果:PCR获得长516bp的片段。Loa22基因去信号肽片段与pGEX-4T-1的重组质粒构建成功。重组质粒经IPTG诱导后能在大肠杆菌中表达Mr45000的融合蛋白。结论:制备了Loa22基因去信号肽片段原核重组表达载体,为钩体新型疫苗的研究奠定基础。AIM： To construct the prokaryotic expression vector of the OmpA membrane protein Loa22 gene excluding signal peptide of Leptospira lai and analyze its clonal expression. METHODS： The experiment was conducted in the Institute of Infection and Immunity of West China Medical Center of Sichuan University between December 2004 and December 2005. The Loa22 gene excluding signal peptide was amplified by polymerase chain reaction （PCR） from genome of Leptospira lai 017 strain. Amplified DNA fragment was subcloned into vector pGEX-4T-1. The masculine clone of recombinant plasmid was screened by double enzyme digestion and PCR. DNA sequence was performed subsequently. Later, Loa22 excluding signal peptide was induced by IPTG to express and be identified by SDS-PAGE. RESULTS： About 516 bp fragment was amplified in Leptospira lai 017 strain. The recombinant plasmid of Loa22 excluding signal peptide and pGEX-4T-1 was constructed and expressed fusion protein （M=45 000） in E. coil JM109 induced by IPTG. CONCLUSION： The successful construction of the prokaryotic recombinant expression vector of Loa22 gene excluding signal peptide provides a basis for exploring the new type vaccine of Leptospira.

关 键 词：钩端螺旋体 OMPA 信号肽 原核表达 

分 类 号：R349[医药卫生—基础医学]