结核分枝杆菌CFP10诊断抗原基因的克隆及序列特性分析

Cloning and sequence analyzing of the CFP10 gene from Mycobacterium tuberculosis

目的扩增结核分枝杆菌培养滤液蛋白10基因(CFP10),并将其克隆至质粒pGEMT中进行核苷酸序列特性分析,为研究结核病候选诊断抗原基因奠定基础。方法以结核菌标准菌株H37Rv为模板,通过PCR技术扩增出结核分枝杆菌CFP10抗原基因,将其重组到pGEM-T载体后进行酶切鉴定及序列测定和生物信息学分析。结果成功扩增出结核分枝杆菌CFP10抗原基因,测序表明该片段开放阅读框由303bp组成,与已发表基因核苷酸序列相比,同源性为100%,推导编码氨基酸序列同源性为100%。结论成功克隆CFP10基因,经DNA测序证实,该片段阅读框完整,为其原核表达及相关研究奠定了基础。

Objective To obtain and analyze characters and homologies of sequence CFP10 gene,and lay bases for screening candidate antigen of Mycobacterium tuberculosis.Method Total RNA was extracted from protoscoles of cysts.The CFP10 gene of Mycobacterium tuberculosis was amplified by PCR and recombined into pGEM-T vector for sequencing and analyzing.Results A DNA sequence with an open reading frame of 303bp was successfully amplified by PCR.Compared with the DNA sequence published,the homologies were 100%,which deduced that the amino acid sequence of CFP10 of Mycobacterium tuberculosis were 100% identities.Conclusion We can make sure that the segment of amplification is CFP10,and its reading frame is integrity.The study laid a good foundation of the prokaryotic expression and research of the gene.