摘要
目的:从结核分枝杆菌基因组克隆Rv3881c基因并重组入pET24b载体,在大肠杆菌中进行融合表达,重组蛋白经纯化后通过Western blot分析初步鉴定其抗原性和特异性。方法:提取H37Rv标准株的基因组DNA,以基因组DNA为模板PCR扩增出Rv3881c基因,连接到pGEMT上,经过筛选鉴定后,将重组子测序;将测序正确的基因双酶切后连接到pET24b载体上,筛选得到重组载体pET24b-48;在BL21菌株中优化表达并采用金属鏊合层析方法纯化蛋白;选取6例结核病阳性临床血清标本和6例健康人血清标本进行Western blot分析。结果:发现从保存的H37Rv菌株克隆得到了读码框完全正确的Rv3881c基因,它在大肠杆菌中可以高效表达,表达量约占细菌总蛋白的20/以上。重组蛋白不与健康人血清反应,可与结核标本血清有较强的免疫印迹反应。结论:重组Rv3881c具有较好的特异性,该抗原有潜力成为检测结核的有效抗原组分之一。
Objective The inclusion of Rv3881c in a prototype serediagnostic test will highly increase assay sensitivity and speciality for M. tuberculosis infection when it is combined with other known immunodominant antigens. To obtain purified recombinant Rv3881c antigen, Rv3881c gene was cloned and inserted into prokaryotic expressing vector pET24b, then efficiently expressed in BL21. Method Primers for Rv3881c gene was designed. DNA fragment encoding Rv3881c antigen was obtained by PCR frozen mycobacterium tuberculosis Genomic DNA and cloned into pGEM-T vectors, then sequenced. DNA fragments encoding Rv3881c were inserted into prokaryotic expressing vector pET24b. The pET24b-48 plasmids were transfered into BL21, then cultured and induced by IPTG to obtain fusion proteins. Recombinant fusion protein with a 6×His tag in the C terminus, is purified by Ni-chelating Sepharese Fast Flow reagent. Antigenicity of rRv3881c was simply evaluated by Western blot through the reactivity with sera from 6 M.tuberculosis patients. Result Rv3881c was sequenced and proved to be correct. The amount of fusion proteins contained Rv3881c antigen and His-tag expressed in BL21 occupy more than 20% of total bacterial proteins. Purified protein can react strongly with 6 serum samples from M.tb patients, but not with serum samlpes from healthy control. Conclusion The results indicate that this kind of recombinant antigen show high speciality to sera of M. tuberclosis patients and might be a candidate of diagnostic antigens.
出处
《实用医学杂志》
CAS
2007年第11期1605-1607,共3页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(批准号:30471528和30471628)。
