牛分枝杆菌广西分离株MPB70基因的克隆及序列分析

利用牛分枝杆菌MPB70基因特异性引物,以2株牛分枝杆菌广西分离株的染色体DNA为模板,扩增MPB70基因,产物经纯化后与载体PMD18-T连接,然后转化至大肠杆菌DH5α。提取转染后大肠杆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DNAstar MegAlign对MPB70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8株牛分枝杆菌的MPB70基因参考序列进行比较。结果显示广西分离株mt359、mt370与已发表的7株参考株序列比较,其核苷酸序列和氨基酸同源性在77.4%~95.9%之间。这一结果表明广西奶水牛分枝杆菌分离株与参考的其他牛分枝杆菌毒株的MPB70基因没有太大的差异,说明牛分枝杆菌分泌蛋白MPB70基因十分保守,从而为检测跟踪毒株的变异,研制牛分枝杆菌亚单位疫苗提供基础。

Cloning and Nucleotide Sequencing Analysis of the MPB70 Gene of Mycobacterium bovis of Reference and Isolate Strains

Based on the secreted protein MPB70 gene sequences of mycobacterium bovis,a pair of specific primers was designed and synthesized for amplification the whole MPB70 genes.Total 2 of DNA extraction from field isolate strains in Guangxi Province were amplification by polymerase chain reaction（PCR）.The PCR products were purified and ligated with PMD18-T vector and the vector was transferred to E.coli DH5α.The plasmid was sequencing after identification by digestion with Hind III,EcoR I and PCR.MPB70 gene nucleotides and predicted amino acids were analysis using DNAStar MegAlign software.The results revealed that the isolate strains mt359 and mt370 had similarity homology between 77.4% to 95.9% with the 8 reference strains from the GenBank.This means that secreted protein MPB70 gene is a very conservative,but also has characteristics of immune genes.It will be good for monitor testing for the mutation.The DNA vaccine with MPB70 gene will develop in the future.