结核分枝杆菌Rv3914抗原的克隆表达与纯化  

Cloning,expression and purification of Rv3914 antigen of M.tuberculosis

作　　者：陈永金 赵俊伟[2] 杨红国 孙战强[2] 余晓丽[3] 张舒林[2] 

机构地区：[1]临沂市胸科医院,山东临沂276034 [2]上海交通大学医学院 [3]湖北省武汉工业学院

出　　处：《中国预防医学杂志》2011年第11期902-905,共4页Chinese Preventive Medicine

基　　金："十一五"国家科技重大专项(2009ZX10004-313);"十一五"国家科技重大专项(2009ZX10003-017);湖北省教育厅科研项目(B20101701)

摘　　要：目的构建结核分枝杆菌Rv3914蛋白的重组质粒,并在大肠埃希菌中表达及纯化,为结核病血清学诊断提供候选抗原。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增Rv3914基因片段,插入到pET-28b(+)载体中,构建pET28b-Rv3914重组质粒,转化大肠埃希菌E.coli BL21plysS(DE3),经IPTG诱导表达后,应用Ni-NTA亲和柱纯化重组蛋白。结果 PCR扩增出Rv3914基因序列,重组质粒经测序并经BLAST分析后发现无点突变。pET28b-Rv3914重组质粒在大肠埃希菌E.coli BL21plysS(DE3)中主要以可溶性形式表达,重组蛋白占细胞蛋白总表达量的30%以上,经Ni-NTA柱纯化后,得到纯度超过90%、相对分子质量约为14.7×103的目的蛋白质。结论高纯度结核分枝杆菌Rv3914重组蛋白的获得为研究结核互补特异性抗原组合在结核病血清诊断中的价值奠定了基础。Objective To investigate the cloning and expression of Rv3914 antigen of M.tuberculosis,thus provide a candidate antigen for TB serodiagnosis.Methods The gene fragment encoding Rv3914 protein was amplified by PCR from the genome DNA of M.tuberculosis H37Rv strain and then inserted into corresponding site of the expression vector pET-28b（＋）.The recombinant plasmid pET28b-Rv3914 was transformed into E.coli BL21（DE3）pLysS strain and induced with IPTG.The recombinant protein was purified by Ni-NTA purification system.Results The inserted sequence of Rv3914 gene fragment was identical to the gene recorded in GeneBank.The expected molecular weight of 14.7 kDa recombinant protein was expressed in E.coli BL21 plysS（DE3） and amounted to more than 30% of total cell proteins.The recombinant protein was purified by Ni-NTA purification system with high purity（more than 90%）.Conclusion The high purity of Rv3914 recombinant protein has potential applications for the specific serodiagnositic research of M.tuberculosis infection.

关 键 词：结核分枝杆菌 Rv3914 克隆 表达 纯化 

分 类 号：R378[医药卫生—病原生物学]