边缘无浆体MSP5基因片段的克隆与原核表达  

CLONE AND PROKARYOTIC EXPRESSION OF MSP5 GENE FOR ANAPLASMA MARGINALE

作　　者：杜凯[1] 李树清[2] 陈志飞[2] 周艳琴[3] 王贵强[3] 王巧全[2] 姚宝安[3] 赵俊龙[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]上海出入境检验检疫局,上海200135 [3]华中农业大学动物医学院,武汉430070

出　　处：《中国兽医寄生虫病》2007年第4期11-15,共5页Chinese Journal of Veterinary Parasitology

基　　金：国家质量监督检验检疫总局项目(2005ZK179);上海市科委标准专项项目(04DZ05017)

摘　　要：目的克隆边缘无浆体MSP5基因,构建重组质粒,并进行表达与鉴定。方法根据GenBank公布的边缘无浆体MSP 5基因序列(AY714547)设计一对引物,无菌颈静脉采集边缘无浆体阳性的牛血,用PCR方法从4血的DNA模板中扩增MSP 5基因582bp的片断。将该片段克隆到原核表达载体pGEX-KG中,构建原核表达载体KG-MSP5,转化BL21,经IPTG的诱导表达蛋白。利用BugBuster GST Bind Purification Kit将其纯化,经Western blotting进行分析。结果重组质粒KG-MSP5,转化BL21,在IPTG的诱导下表达大小约46 kPa蛋白。western blotting分析表明其具有很好的免疫原性。结论本研究为边缘无浆体的血清学诊断试剂盒的研制奠定了基础。Objective The cloned partial MSP5 of Anaplasma marginale was inserted into pGEX-KG to construct a prokaryotic expression vector. This recombinant was then transformed into Escherichia coli BL21 in order to obtain the protein as the foundation for further study. Methods Using a pair of specific primers designed according to the relevant nucleotide sequence （AY714547） from GenBank, the MSP5 gene of Anaplasma marginale was amplified with PCR method. The PCR product was cloned into pGEX-KG to construct a prokaryotic recombinant plasmid KG-MSPS. Then the recombinant plasmid KG-MSP5 was transformed into E. coli BL21. The target protein was purified by BugBuster transformed into E. coli BL21. The target 46kD protein was found in the IPTG-inducible product and finally purified by BugBuster GST Bind Purification Kit. Western blotting analysis proved the recombinant protein had good reactive ability against Anaplasma marginale positive serum. Conclusion The results lay foundation for the development of a detecting Kit against Anaplasma marginale.

关 键 词：边缘无浆体 MSP5 原核表达 纯化 

分 类 号：S852.64[农业科学—基础兽医学]