摘要
目的以梅毒螺旋体(Tp)外膜蛋白基因Gpd及细胞因子佐剂IL-2为目的基因,融合构建原核表达重组体pET28a/Gpd-IL-2,表达鉴定并进行免疫活性的初步分析,为进一步开展Tp疫苗动物实验打下基础。方法定向克隆构建原核表达重组体pET28a/Gpd-IL-2进行诱导表达;Ni亲和层析纯化重组融合蛋白后,Western blot检测其免疫反应性;免疫新西兰兔评价重组蛋白的免疫原性。结果成功构建原核表达重组体pET28a/Gpd-IL-2,表达鉴定及纯化获得相对分子量约为60 kDa的融合蛋白;Western blot检测其能与梅毒阳性血清发生特异性结合,说明两融合基因之间的表达相互没受到太大影响;纯化的Gpd-IL-2重组蛋白免疫新西兰兔能诱导其产生特异性免疫应答,ELISA检测免疫血清中特异性抗体滴度在1∶6 400以上。结论 Gpd-IL-2融合基因重组蛋白具有良好的免疫活性,这为进一步开展Tp疫苗动物实验条件的优化打下一定的基础。
Objective Expression and Identification of Gpd-IL-2 recombinant protein of Treponema pallidum and its immuno-competence analysis.Methods The target genes encoding Gpd and IL-2 were subsequently cloned into the expression plasmid pET28a（＋）,and expressed in E.coli Rosseta.Its immunogen was analysed by Western blotting and ELISA after New Zealand White rabbits were immunized by three subcutaneous inoculations of Tp Gpd recombinant protein. Results The recombinant prokaryotic expression vector pET28a/Gpd-IL-2 was successfully constructed and a purified fusion protein with molecular weight of about 60 kDa was expressed and identified.Western blotting proved that the fusion recombination protein can specifically react with Tp positive sera.Specific humoral response were elicited after introducing recombinant protein in Zealand rabbit and the specific antibody titer was above 1∶ 6400 detected by indirect ELISA.Conclusion The expressed Gpd-IL-2 fusion recombinant protein showed excellent immuno-competence,and the results lay the foundation for the research on T.pallidum vaccine optimization.
出处
《中南医学科学杂志》
CAS
2012年第2期131-135,共5页
Medical Science Journal of Central South China
基金
湖南省科技厅重点课题(2010FJ2008)
湖南省科技厅项目(2011tt2014)
湖南省自然科学省市联合基金(11JJ9023)
湖南省自然科学基金青年基金(11JJ4076)
