摘要
目的 构建结核分枝杆菌分泌蛋白MPT 64的重组质粒,并在大肠杆菌中表达及纯化和复性重组蛋白.方法 用PCR方法从结核分枝杆菌H37Rv基因组中扩增出mpt64基因片段,插入PKRX-T载体中,再亚克降到表达载体PET30a中,在大肠杆菌中表达;应用组氨酸标签（His-Tag）纯化重组蛋白;在PBS中通过半透膜恢复重组蛋白的天然结构.结果 构建了含MPT64重组质粒的大肠杆菌工程菌,并以包涵体形式表达重组蛋白MPT 64,其占细胞总蛋白的20%～30%;重组蛋白变性后经镍柱纯化,其纯度达90%以上;通过半透膜完全恢复重组蛋白的天然结构.结论 具有天然结构和高纯度的重组融合蛋白有可能成为有效的结核病血清学诊断的候选抗原.
Objective To construct the recombinant plasmid of secreted protein MPT 64 of mycobacterium tuberculosis and to express, purificate and renatruate fusion protein MPT 64. Method the gene coding MPT 64 protein was amplified by polymerase chain reaction （PCR） from genome of mycobacterium tuberculosis H37Rv , then cloned into PKRX - T - easy and recloned into expression vecter PET30a and expressed fusion protein MPT 64 in E.coli BL21 （DE3） .It was purificated by his - tag of the fusion protein MPT 64 and renaturated by semipermeable membrane. Results The recombinant MPT 64 protein existed in inclusion bodies of E.coli.and amounted to 20% - 30% of totle cell protein.The inclusion bodies was fully renaturated gy semipermeable membrane. Conclusion The recombinant protein of purification and renaturation may be a potential candidate to be a serodiagnostic reagent.
出处
《中国预防医学杂志》
CAS
2007年第2期113-115,共3页
Chinese Preventive Medicine
基金
国家卫生专项工作经费
