【摘要】 通过对牛环形泰勒虫Tams1基因的不同片段进行克隆表达,以期得出该基因疏水区对蛋白表达的影响及筛选的目的蛋白作为ELISA抗原的特异性。利用3对不同的特异性引物对Tams1基因的三段区域(全长基因、无N端及C端疏水区、仅缺乏N端的疏水区)进行克隆,最终构建三种重组表达载体。经电泳分析筛选获得可溶性表达重组质粒,目的蛋白经亲和层析纯化后进行Western-blot分析。结果显示,在37℃条件下,经终浓度为1mmol/L IPTG诱导表达4h后,无疏水区的靶重组质粒表达出53ku的可溶性蛋白,其表达量达1.39mg/mL;而含全长基因或C端疏水区的质粒分别表达55ku、56ku的包涵体蛋白。Western-blot分析结果表明,筛选得到的可溶性蛋白能被环形泰勒虫阳性血清特异性识别,具有良好的反应原性。结果表明,该蛋白可作为潜在的进行牛环形泰勒虫病流行病学调查的候选抗原。更多还原

【Abstract】 Effects of Tams1gene hydrophobic domains on its expression and specificity of screened protein used as ELISA antigen were studied by cloning and expressing the different gene fragments.Three fragments including the complete gene,the complete gene lacking N-terminal and C-terminal hydrophobic domain,and the gene fragment without the sequences coding for N-terminal hydrophobic domains of Tams1gene were amplified by PCR using three sets of specific primers.Then three recombinant plasmids were constructed and finally 3high soluble expression recombinant plasmids were screened and the expressed protein was analyzed by SDS-PAGE and Western-blot.In result,the recombinant plasmid without the sequences encoding for both hydrophobic domains could express 53ku soluble protein when induced with 1mmol/L IPTG for 4hat 37℃,and its expression level reached 1.39mg/mL.Soluble expression content of the recombinant plasmid with the complete gene or C-terminal hydrophobic domain were few and were mainly 55ku and 56ku proteins in a form of inclusion bodies,respectively.Western-blot analysis showed that the soluble protein could be specifically recognized by the positive serum against Theileria annulata,so it had good reactionogenicity.The result showed that acquired soluble protein could become a useful tool to survey the epidemiology of Theileria annulata.更多还原