肺炎支原体双蛋白多抗原表位表达载体的构建  

Construction of expression vector of specific two-protein multi-antigen epitope gene of Mycoplasma pneumoniae

作　　者：卢可新[1] 赵芝娜[1,2] 刘宝山[1,3] 赵晨超[1] 王舰[1] 王桂珍[1] 

机构地区：[1]中国医科大学病原生物教研室,辽宁沈阳110001 [2]沈阳药科大学,辽宁沈阳110016 [3]沈阳农业大学,辽宁沈阳110866

出　　处：《中国微生态学杂志》2011年第9期810-812,815,共4页Chinese Journal of Microecology

基　　金：辽宁省教育厅基金项目(2004D226)

摘　　要：目的构建肺炎支原体(Mp)双蛋白多特异抗原表位表达载体,提高重组蛋白抗原的敏感性。方法应用生物信息学方法筛选Mp P116粘附蛋白抗原表位序列,PCR点突变技术获取P116蛋白基因片段,与pMD-T载体重组,转入大肠埃希菌JM109,通过限制性酶切图谱和基因序列分析鉴定重组质粒。酶切回收P116基因片段与pGEX 6P-1-P1 DNA重组,转入大肠埃希菌JM109菌株。用Glutathione Sepharose 4B纯化重组蛋白,SDS-PAGE分析表达产物的相对分子量,用Mp免疫血清进行免疫印迹试验,鉴定重组蛋白的免疫原性。结果 PCR点突变扩增Mp黏附蛋白P116的基因片段为597 bp,该基因片段与已知的基因库序列分析比较,除两个突变位点由UAG突变为UGG外,其余核苷酸序列同源性为100%。SDS-PAGE分析多表位重组蛋白相对分子质量(Mr)为77.8 kDa。免疫印迹结果显示,Mp兔多价血清能与纯化的78KDa的重组蛋白发生免疫反应。结论本研究成功构建了Mp双蛋白多表位的表达载体。该表达载体表达的重组蛋白具有Mp特异的免疫反应性。重组蛋白的敏感性有待进一步鉴定。Objective To construct the specific two-protein multi-antigen epitope expression vector of Mycoplasma pneumoniae（Mp） for enhancing the sensitivity of the recombinant protein antigen.Method Antigen epitope sequences of Mp adhesion protein P116 was screened by bioinformatics method.P116 protein gene was obtained by PCR point mutations technology and recombined with pMD-T vector and then transformed into E.coli JM109.Recombinant plasmid was identified and analyzed by restriction enzyme mapping and gene sequence.The P116 fragment was purified and recovered by enzyme digestion and then linked with pGEX6P-1-P1 DNA fragment to produce a recombinant DNA which was transformed into E.coli JM109 strain.Recombinant protein was purified with Glutathione Sepharose 4B.Molecular weight and immunogenicity of recombinant protein was analyzed relatively by SDS-PAGE and immunoblotting.Result The P116 gene fragment amplified by PCR was 597 bp.And compared with the known gene sequence in the GenBank,except for the two mutations from the UGG to UAG,the other nucleotide sequence of the gene was 100% matched.SDS-PAGE analysis showed that the molecular weight（Mr） of the multi-epitope recombinant protein was 77.8 kDa.Western blot analysis showed this recombinant protein could react with Mp polyvalent rabbit serum.Conclusion This study successfully constructed the expression vector of two-protein specific multi-antigen epitope of Mp,and the recombinant protein of this vector possesses specific immune response with Mp.The sensitivity of the recombinant protein will be identified and evaluated further.

关 键 词：肺炎支原体 多抗原表位表达载体 基因克隆 

分 类 号：R375[医药卫生—病原生物学]