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目的对比不同方式克隆表达鼠疫耶尔森氏菌caf1M蛋白的存在状态及免疫学活性。方法分别选择3种不同载体pET32a(+)、pGEX4t-1、pGBTNH,克隆表达鼠疫caf1M蛋白;并以鼠疫菌免疫血清分别检测其免疫学活性。结果成功构建了4个重组质粒,分别是含去除信号肽编码序列caf1M基因的3个质粒载体,及1个含有完整caf1M基因的pGEX4t-1表达质粒;4个质粒经诱导均在大肠杆菌中得到高效表达;存在状态分析表明,含有去除信号肽编码序列caf1M基因的pGEX4t-1表达的重组蛋白以可溶方式存在,其余3种以包涵体形式存在;重组蛋白与鼠疫菌免疫血清的Western blot分析表明,除pGBTNH表达的蛋白存在非特异反应外,其余3种重组蛋白都发生特异性反应。结论成功克隆表达了4种鼠疫菌caf1M蛋白,其中3种具有良好的特异性免疫反应性;信号肽序列及载体都对重组caf1M蛋白的表达有影响。
Objective To compare the existence and immunological activities of different ways of cloning and expressing caf1M protein of Yersinia pestis. Methods Three different vectors, pET32a (+), pGEX4t-1 and pGBTNH, were selected respectively to express the caf1M protein of plague. The immunological activity of P.faecalis was tested by immunological sera. Results Four recombinant plasmids were successfully constructed, including three plasmid vectors containing the caf1M gene with the coding sequence of the signal peptide and one pGEX4t-1 expression plasmid containing the complete caf1M gene. The four plasmids were all induced in E. coli The results of existent state analysis showed that recombinant protein containing pGEX4t-1 with caf1M gene with signal peptide coding sequence existed in soluble form and the other three existed as inclusion body. Western blot analysis of the recombinant protein with immune virus of Y. pestis The results showed that all the three recombinant proteins reacted specifically except pGBTNH. Conclusion Four caf1M proteins of Y. pestis were successfully cloned and expressed, of which three had good specific immunoreactivity. The signal peptide sequence and vector all had effects on the expression of recombinant caf1M protein.