牛分枝杆菌FixB、HspX和MPB83基因的原核表达及鉴定  

Prokaryotic expression and identification of FixB、HspX and MPB83 genes from Mycobacterium bovis

作　　者：陈爽[1,2] 许芳[2,3] 张林波[1] 张喜悦[1,2] 范伟兴[2] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]甘肃农业大学动物医学院,兰州730070

出　　处：《黑龙江畜牧兽医》2017年第6期15-19,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：科技基础性工作专项(2012FY111000);现代农业(奶牛)产业技术体系建设项目(CARS37)

摘　　要：为了给原核表达牛分枝杆菌Fix B、Hsp X和MPB83基因并纯化Fix B、Hsp X和MPB83蛋白,进一步研究它们在牛结核病诊断中作为特异性抗原的诊断价值及应用奠定基础,试验用PCR方法从牛结核分枝杆菌AF2122/97基因组中扩增出Fix B、Hsp X和MPB83基因片段,构建p ET-28a-Fix B、p ET-28a-Hsp X和p ET-28a-MPB83重组质粒,克隆到DH5ɑ感受态细胞,提取测序正确的阳性重组质粒,转化BL21(DE3)感受态细胞,用1 mmol/L IPTG于37℃诱导表达,SDS-PAGE分析目的蛋白的表达形式,Western-blot分析鉴定并用Ni-NTA层析柱纯化蛋白。结果表明:成功扩增了大小依次约为957 bp、435 bp和663 bp的Fix B、Hsp X和MPB83基因,将其连接p ET-28a原核表达载体,原核表达的Fix B、Hsp X和MPB83重组蛋白均以包涵体形式存在,分子质量依次约为37 ku、21 ku和28 ku。经Western-blot鉴定表达产物为Fix B、Hsp X和MPB83重组蛋白,Ni-NTA成功洗脱出目的蛋白。说明试验成功构建了牛分枝杆菌Fix B、Hsp X和MPB83基因的原核表达载体,并纯化了Fix B、Hsp X和MPB83重组蛋白,可用于进一步的应用研究。To express the genes FixB, HspX and MPB83 from Mycobacterium bov/s and further investigate FixB, HspX and MPB83 proteins as specific antigens for the diagnostic value and application, specific primers for FixB, HspX and MPB83 genes were designed, respectively, and then the genes FixB, HspX and MPB83 were amplified from Mycobacterium bovis AF2122/97 strain genome by PCR; the PCR products were cloned into the prokaryotic vector pET -28a, resulting in generation of recombinant plasmids pET- 28a -FixB, pET- 28a -HspX and pET- 28a- MPB83; after sequencing, the recombinant plasmids were transformed respectively into E. coli BL21 （DE3） for expression with 1 mmol/L IPTG induction at 37 ~C. The expression form of target proteins were analyzed by SDS - PAGE, and then recombinant.proteins were detected by using Western -blot ; the recombinant proteins were purified by using Ni- NTA column. The results showed that FixB, HspX and MPB83 genes were successfully cloned into pET28a prokaryotic expression vector, whose the sizes were about 957 bp, 435 bp and 663 bp, re- spectively. The recombinant proteins FixB, HspX and MPB83 are expressed as form of inclusion body in E. coli BL21 （ DE3 ）, and their molec- ular weight were 37 ku, 21 ku, and 28 ku, respectively; the recombinant proteins FixB, HspX and MPB83 were confirmed by Western -blot, and were successfully purified by using Ni - NTA. These data indicated that recombinant plasmids pET - 28a - FixB, pET - 28a - HspX and pET -28a - MPB83 were successfully constructed, and the expressed recombinant proteins were purified, respectively, which will provide ma- terials for further research.

关 键 词：牛分枝杆菌 FixB HSPX MPB83 原核表达 纯化 

分 类 号：S852.618[农业科学—基础兽医学]