牛分支杆菌广西分离株Hsp 65基因的克隆及序列分析

Cloning and Sequence Analysis of the Hsp 65 Gene of Mycobacterium bovis of Reference and Isolate Strains

利用牛分支杆菌Hsp 65基因特异性引物,对2株牛分支杆菌广西分离菌株进行PCR扩增,产物经纯化后与载体pMD-18连接,然后转染大肠埃希菌DH5α。提取转染后大肠埃希菌的质粒进行双酶切和PCR鉴定,鉴定为阳性的质粒进行序列测定。测序后通过序列分析软件DNA Star MegAlign对Hsp 65基因核苷酸序列及推导的氨基酸序列进行分析,并与GenBank上已发表的牛分支杆菌的Hsp 65基因进行比较。结果显示,广西分离株mt359、mt370与已发表的7株参考株序列,其核苷酸序列同源性在98.7%～100%之间,推导的氨基酸序列同源性在98.4%～100%之间。表明广西分离的菌株与参考的其他牛分支杆菌菌株的Hsp 65基因差异不大,说明牛分支杆菌分泌蛋白Hsp 65基因十分保守,从而为检测跟踪菌株的变异,研制牛分支杆菌亚单位基因疫苗奠定基础。

Based on the published hot shock protein 65（Hsp 65） gene sequences of mycobacterium bovis,a pair of specific primers were designed and synthesized for amplification the whole Hsp 65 genes.Total 2 of DNA extracts from field isolate strains in Guangxi were amplification by polymerase chain reaction（PCR）.The PCR products were purified and ligated in PMD18-T vector and the vector was transferred to E.coli DH5α.The plasmid was sequenced after identification by digestion with HindⅢI,EcoRⅠ and PCR.Hsp65 gene nucleotides and predicted amino acids were analysis using DNAstar MegAlign software.The results revealed that the isolate strains mt359,and mt370 had a homology in nucleotide sequence from 98.7% to 100% with the 7 standard reference strains from GenBank.The homology in deduced amino acid sequence was between 98.4% to 100%.This means that secreted protein Hsp 65gene is very conservative,but also has characteristics of immune genes.It will be good for monitor testing for the mutation.The DNA vaccine with Hsp65 gene will develop in the future.