摘要
目的 对结核杆菌Ag85A抗原基因进行克隆、表达与纯化 ,为研制新型结核杆菌血清学诊断试剂奠定基础。方法 以结核杆菌H37Rv株基因组DNA为模板 ,经PCR扩增Ag85A抗原基因成熟肽编码区 ,定向克隆入原核表达载体pGEX1 λT中日本血吸虫谷胱苷肽硫转移酶 (GST)编码区下游构建重组质粒 ,经DNA测序鉴定后 ,转化大肠杆菌JM10 9株 ,IPTG诱导表达 ,采用GST蛋白纯化试剂盒进行重组融合蛋白纯化 ,SDS -PAGE和Westernblot鉴定重组表达产物。结果 PCR扩增获得了约 90 0bp的目的基因片断 ,定向克隆入原核表达载体pGEX1 -λT后 ,对重组质粒进行DNA测序发现 ,插入片段与GenBank中结核杆菌Ag85A抗原基因成熟肽编码区同源性为 99 .8%。重组质粒转化大肠杆菌后 ,经IPTG诱导表达 ,纯化的融合蛋白相对分子质量为 5 80 0 0 ,Westernblot检测该融合蛋白能与兔抗结核杆菌多价抗血清发生反应。结论 本研究为研制结核杆菌血清学检测试剂盒及相关分子免疫学研究奠定了基础。
Objective To clone and express Mycobacterium tuberculosis Ag85A antigen gene,purify th e expressed product and lay a foundation of developing novel serological diagnos tic kit for mycobacterium.Methods The gene fragment encoding mature peptide of M.tuberculosis Ag85A were ampli fied by PCR using the genome DNA of H37Rv strain as a template and inserted down stream to the encoding region of Schistosoma japonicum glutathione S-transf erase (GST) of prokaryotic expression vector pGEX1-λT.The constructed recombin ant plasmid was identified by DNA sequencing and transformed to E.coli JM109 strain for expression under induction of IPTG.The expressed fusion protein was purified by GST purification kit and identified by SDS-PAGE and Western blot.Results A goal gene fragment at length of about 900 bp was amplified by PCR and inserted into expression vector pGEX1-λT.The DNA sequencing result of recombinant plas mid proved that the homology of inserted fragment to the gene encoding mature pe ptide of M.tuberculosis Ag85A antigen,reported in GenBank,was 99 8%.A fusio n protein with relative molecular weight of 58 000 was expressed and recognized by rabbit anti-M.tuberculosis polyclonal antibody.Conclusion The fusion expression of M.tuberculosis Ag85A and S.japonicum GST laid a foundation of developing serological diagnostic kit for mycobacterium and relat ed study in molecular immunology. [
出处
《中国生物制品学杂志》
CAS
CSCD
2005年第2期101-103,共3页
Chinese Journal of Biologicals
基金
四川省教育厅自然科学重点项目 (2 0 0 3A0 67) .
