边缘无浆体膜表面蛋白1a(MSP1a)的克隆与原核表达  

Cloning and prokaryotic expression of MSP1a protein-encoding gene of Anaplasma marginale

作　　者：姜海芳[1] 倪宏波[1] 王春仁[1] 宫大庆[1] 邵光喜[1] 周玉龙[1] 崔玉东[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《黑龙江畜牧兽医》2010年第11期13-15,共3页Heilongjiang Animal Science And veterinary Medicine

基　　金：吉林省科技发展计划项目(20100220);兽医生物技术国家重点实验室开放课题基金项目(SKLVBF200807);黑龙江省科技厅实验动物青年基金项目(PC07S09)

摘　　要：为了给牛边缘无浆体的诊断和基因工程疫苗的研制提供理论依据,试验以牛边缘无浆体基因组DNA为模板,采用PCR技术对其膜表面蛋白1a(MSP1a)基因进行扩增,将扩增产物转入克隆载体和表达载体中进行克隆和表达,用重组表达质粒转化大肠杆菌BL21(DE3),利用免疫印迹分析鉴定MSP1a基因表达产物的免疫活性。结果表明:MSP1a基因PCR扩增产物与GenBank上的PR1株MSP1a基因的序列同源性达98.3%,编码氨基酸的同源性为98.7%;将该基因亚克隆入原核表达载体pET-28a中,构建了重组原核表达载体;将其转化到DH5α大肠杆菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为49 ku;经SDS-PAGE电泳和免疫印迹检测,MSP1a基因在表达载体中得到高效表达,且表达产物具有免疫活性。To provide the theoretical basis for diagnosis on Anaplasma marginale（A.marginale） development of genetic engineering vaccine.A pair of specific primers were designed according to the nucleotide sequence of the published MSP1a gene of A.marginale.The MSP1a gene was amplified by PCR based on the genomic DNA template of A.marginale,and then was cloned into pMD18-T vector and subsequently identified by restriction endonuclease digestion,PCR and sequencing.In consequence,the homology sequence at a nucleotide level was 98.3% compared with MSP1a gene from A.marginale PR1 strain based on the GenBank and the amino acid similarity was 98.7 % correspondingly.The MSP1a gene was subcloned into PET-28a vector to construct a prokaryotic expression vector pET-28a/MSP1a and transformed into BL21（DE3）.The recombinant plasmid PET-32a/MSP1a was induced in E.coli by using IPTG.Western-blot showed that the molecular weight of expressed recombinant fusion protein was 49 ku.The result of SDS-PAGE electrophoresis and western blot indicated that the MSP1a gene of A.marginale had high level expression in expression vector,and was able to be recognized by the rabbit anti-A.marginale positive serum.

关 键 词：边缘无浆体 MSP1a基因 克隆 原核表达 

分 类 号：S852.64[农业科学—基础兽医学]