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克隆细粒棘球绦虫EgA31抗原基因,构建pET30a-EgA31原核表达载体,并在大肠埃希菌宿主系统中表达EgA31重组蛋白,对表达产物进行SDS-PAGE分析。从细粒棘球绦虫成虫组织中提取总RNA,反转录生成cDNA,以此cDNA为模板RT-PCR克隆获得EgA31抗原基因,将其克隆至pUCm-T载体,测序确定其正确性。利用定向克隆技术将EgA31抗原基因片段克隆至原核表达质粒pET30a上,转化E.coliDH5α,根据选择标记的卡那霉素抗性基因筛选到阳性克隆,通过PCR分析和酶切鉴定筛选出阳性克隆。IPTG初步诱导和表达pET30a-EgA31重组蛋白,SDS-PAGE电泳检测,并经凝胶图像分析确定目的蛋白的表达水平。测序表明选取的pET30a-EgA31阳性克隆均为正确连接EgA31抗原基因的重组质粒。经IPTG诱导后重组蛋白得到成功表达,在相对分子量约为31kDa处有表达条带,表达量约占菌体总蛋白质的26%。成功克隆并构建了pET30a-EgA31原核表达质粒,初步诱导表达出EgA31重组蛋白,为进一步研究其免疫特性奠定了基础。
The EgA31 antigen of Echinococcus granulosus was cloned, and the prokaryotic expression vector pET30a-EgA31 was constructed. The recombinant protein of EgA31 was expressed in Escherichia coli host system, and the expressed product was analyzed by SDS-PAGE. The total RNA was extracted from adult worms of Echinococcus granulosus and cDNA was reverse transcribed. The cDNA of EgA31 was cloned by RT-PCR and cloned into pUCm-T vector. The correctness of sequencing was confirmed by sequencing. The gene fragment of EgA31 was cloned into the prokaryotic expression plasmid pET30a using directional cloning technique. The recombinant plasmid was transformed into E. coli DH5α. Positive clones were screened by selecting kanamycin resistance genes. The positive clones were screened by PCR and restriction enzyme digestion. IPTG primary induction and expression of pET30a-EgA31 recombinant protein, SDS-PAGE electrophoresis detected by gel image analysis to determine the expression level of the target protein. Sequencing showed that the selected pET30a-EgA31 positive clones were both recombinant plasmids correctly linked with the EgA31 antigen gene. After induced by IPTG, the recombinant protein was successfully expressed, with a relative molecular weight of about 31kDa expression bands, the expression amount of about 26% of the total bacterial protein. The prokaryotic expression plasmid pET30a-EgA31 was successfully cloned and constructed, and the recombinant protein of EgA31 was initially induced to lay the foundation for further study of its immunological properties.