结核分枝杆菌Ag85A真核表达质粒的构建与鉴定

CONSTRUCTION OF A EUKARYOTIC EXPRESSION VECTOR OF MYCOBACTERIUM TUBERCULOSIS AG85A

目的 :构建结核分枝杆菌Ag85A重组真核表达质粒 ,为DNA疫苗的研究提供靶基因。方法 :采用聚合酶链反应 (PCR)方法从结核分枝杆菌H37Rv基因组DNA中扩增出结核杆菌Ag85A分泌性蛋白的基因 ,应用T -A克隆技术 ,直接与pUCm -T载体连接 ,转化大肠杆菌JM1 0 9(E .coliJM1 0 9) ,蓝白筛选 ,阳性克隆经酶切鉴定和DNA测序证实基因碱基无误后 ,用NheⅠ和XbaⅠ双酶消化 ,回收的小片段与用NheⅠ和XbaⅠ消化的真核表达质粒pCI-neo连接 ,转化E .coliJM1 0 9,筛选阳性克隆酶切鉴定。结果 :经酶切鉴定 ,证实结核分枝杆菌重组真核表达质粒pCI-Ag85A构建正确。结论 :结核分枝杆菌真核表达质粒pCI-Ag85A构建成功 。

Objective:To construct a recombinant eukaryotic expression plasmid containing the gene encoding the protein Ag85A of mycobacterium tuberculosis H 37 Rv strain.Methods:The full length of TB.Ag85A gene from the genome of M.tuberculosis H 37 Rv was amplified by PCR technique and cloned into pUCm-Tvector with the T-A cloning technique. Then the recombinant plasmid was digested with NheⅠ and XbaⅠ and subcloned into plasmid pCI-neo.Results:By restriction endonuclease digestion and DNA sequencing,it was confirmed that the recombinant eukaryotic expression plamid (pCI-Ag85A)has been successfully constructed. Conclusion:The recombinant plasmid pCI-Ag85A has been constructed. The result serves as a basis for further studies on precaution and therapy of tuberculosis.