日本血吸虫P14钙结合蛋白样蛋白基因真核表达载体的构建、表达和鉴定

摘要

目的 从日本血吸虫成虫cDNA中扩增出钙结合蛋白样蛋白SjP14编码基因,并亚克隆至真核表达载体pcDNA3.1(+),制备重组分子疫苗.方法 提取日本血吸虫成虫总RNA,逆转录cDNA,设计引物常规PCR法扩增出SjP14编码基因,通过线性T克隆载体pGEM-T连接,亚克隆至真核表达载体pcDNA3.1(+),经转染至Hela细胞中表达,表达产物经Western blot鉴定.结果 PCR扩增产物与预期大小相一致,pGEM-T-SjP14及pcDNA3.1(+)-SjP14分别经双酶切后均获得一特异性基因片段,表达产物经Western blot鉴定分子量约为38ku.结论 成功构建了日本血吸虫钙结合蛋白样蛋白真核表达载体,并获得了表达产物.%Objective SjP14 calponin-like protein gene from cDNA of Schistosoma japonicum adult worms were amplified and subcloned into eukaryotic expression vector pcDNA3. l( +) for a new vaccine candidate. Methods The total RNA of S. japonicum was extracted to prepare for cDNA by RT-PCR. The SjP14 encoding gene was amplified. The DNA fragment was subcloned into eukaryotic expression vector pcDNA3. 1( + )following the insertion and amplification in pGEM-T. The recombinant plasmid was transfected into Hela cells and expression products was identified by Western blot. Results The size of PCR product was approximately 1 070 bp, and the inserts of pGEMT-SjP14 and pcDNA3. 1( + )-SjP14 digested with BamH Ⅰ and Xho Ⅰ which were all the same length as PCR product. The recombinant DNA with SjP14 could be expressed in Hela cells and the molecular weight was approximately 38 ku. Conclusion The eukaryotic expression vector carring sjP14 gene has been established and the expression products has been obtained and identified.