摘要
为获得区别结核感染状态的血清学鉴别诊断试剂的抗原,根据GenBank中登录的结核杆菌H37Rv的ACR基因序列设计1对引物,以热灭活的结核杆菌H37Rv菌悬液为模板,PCR扩增得到ACR基因DNA片段。将扩增片段克隆于pGM-T载体中,得到载体pGM-T-ACR。分别将pET32a质粒和pGM-T-ACR质粒用限制性内切酶BamHⅠ和XhoⅠ双酶切,并将纯化的ACR基因亚克隆至pET32a中,获得重组表达质粒pET32a-ACR。将其转化E.coliBL21IPTG诱导后,经SDS-PAGE、westernblot分析鉴定,可见约34ku的外源蛋白带。表明ACR基因在大肠杆菌中得到了表达。用Ni-NTAAgarose试剂盒进行蛋白纯化,获得纯化的ACR融合蛋白。
To obtain the antigen for the development of method to differentiate Mycobacterium tuberculosis （MTB） infection. A pair of primers specific to the ACR gene of MTB was designed and synthesized according to the nucleotide sequences of MTB H37Rv strain published in GenBank. ACR gene was amplified by PCR from inactivated MTB. The PCR product ofACR gene was cloned into the pGM-T vector and verified by sequencing. The ACR gene was subcloned into pET32a to construct recombinant plasmid of pET32a-ACR for expression. The resultant pET32a-ACR was transtbrmed into E.coli BL21 cells and induced by IPTG. The SDS-PAGE and western blot analysis indicated that the recombinant protein was about 34 ku. The recombinant protein was purified with Ni-NTA Agaros.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第7期528-531,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国际科技合作项目基金(2006DFA33740)
石河子大学科研基金(ZRKX200726)
关键词
结核分枝杆菌
ACR
原核表达
Mycobacterium tuberculosis
ACR
prokaryotic expression
