摘要
目的原核表达有免疫学活性的重组caf1M蛋白(rCaf1M),并以此建立检测鼠疫抗体的辅助诊断方法。方法将去掉信号肽编码序列的caf1M基因片段与载体pGEX4t-1通过NcoⅠ和XhoⅠ双酶切位点进行连接,构建重组质粒caf1M-pGEX4t-1;将caf1M-pGEX4t-1转化入E.coliBL21(DE3)中诱导表达;表达产物rCaf1M经亲和层析进行纯化;以纯化rCaf1M及天然F1抗原喷制鼠疫抗体双检测NC膜,并以浙江各6份F1抗体阳性及阴性人血清标本进行应用评价。结果含有重组质粒caf1M-pGEX4t-1的BL21,经诱导产生了分子量约为55×103的rCaf1M融合蛋白;rCaf1M融合蛋白与鼠疫菌免疫血清有较好反应;在浙江人血清标本的检测中,rCaf1M与F1抗体阳性者有明显反应,与F1抗体阴性者无反应。结论本研究制备的rCaf1M,与鼠疫菌免疫血清具有良好的免疫反应性,该rCaf1M可用于鼠疫辅助免疫学检测。
Objective To get recombinant Cafl M protein （rCaflM） being of immunological activity, and construct assistant diagnosis of plague antibody. Methods The caflM gene with removed signal peptide gene was connected with plasmid pGEX4t-1 by double-digested sites of Ncol and XhoI. Recombinant plasmid caflM-pGEX4t-lwas transformed into E. coli BL21 （DE3） and induced to express rCaflM. Expression products of rCaflM were purified by affinity chromatography. The dual detection NC film of plague antibody was constructed with purified rCaflM and natural F1. Six sera which were F1 antibody positive and negative respectively of Zhejiang province were tested with above NC film. Results 55 × 10^3 rCaflM fusion protein was expressed by BL21strains containing caflM-pGEX4t-1. The rCaflM could react with plague anti-sera from rabbit and human. All 6 F1 antibody positive sera of Zhejiang province could react clearly with rCaflM, whereas another 6 FI antibody negative sera couldn＇t. Conclusion The rCaflM developed by this study is of good immunoreactivity with plague anti-serum, and it can be used to serological ussistant examination of plague.
出处
《疾病监测》
CAS
2010年第1期60-64,共5页
Disease Surveillance
基金
国家高技术研究计划项目(2006AA2Z4A7)~~
