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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 express immunologically active recombinant caf1M protein (prokaryotic expression vector) (rCaf1M) in prokaryotic cells and to establish an auxiliary diagnostic method for the detection of plague antibodies. Methods The caf1M gene fragment with the signal peptide coding sequence removed was ligated with the pGEX4t-1 vector to construct the recombinant plasmid caf1M-pGEX4t-1 by NcoⅠand XhoⅠ restriction enzymes. The caf1M-pGEX4t-1 was transformed into E.coli BL21 (DE3 ). The expression product rCaf1M was purified by affinity chromatography. The purified NCF1M and native F1 antigen were used to detect the NC membrane by double antibody detection. The positive and negative serum samples from 6 F1 positive and negative human serum samples from Zhejiang were used for evaluation. Results The recombinant plasmid caf1M-pGEX4t-1 BL21 was induced to produce a rCaf1M fusion protein with a molecular weight of about 55 × 103. The rCaf1M fusion protein reacted well with the Y. pestis immune serum. In the detection of Zhejiang human serum samples, rCaf1M There was a significant reaction with F1 antibody positive and no reaction with F1 antibody negative. Conclusion The rCaf1M prepared in this study has good immunoreactivity with the Y. pestis immune serum and the rCaf1M can be used in the immunization of the plague.