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目的对旋毛虫新生幼虫期特异性T668cDNA进行表达及鉴定,以获得基因工程抗原。方法应用PCR技术,从pBK CMV T668重组质粒中扩增到不含信号肽序列的T668cDNA片段,将目的基因克隆于原核表达载体pET28a(+),构建pETT668重组质粒,再将其转化到E.coli表达菌株DE3感受态细胞中,经IPTG诱导表达,以SDS PAGE鉴定表达产物。结果pETT668表达蛋白的分子质量单位为49ku,且随诱导时间的延长蛋白表达量逐渐增加,诱导5h时表达量达到高峰;薄层扫描结果显示,目的蛋白占菌体总蛋白的34.6%。Western blotting检测显示,表达蛋白可被感染旋毛虫家猪血清所识别。结论旋毛虫新生幼虫期特异性T668基因在大肠埃希菌中获得高效表达,且表达蛋白具有良好的抗原性。
Objective To express and identify the specific T668 cDNA of Trichinella spiralis larvae in order to obtain genetic engineering antigens. Methods The T668 cDNA fragment without signal peptide sequence was amplified by PCR from the recombinant plasmid pBK CMV T668. The target gene was cloned into the prokaryotic expression vector pET28a (+) to construct the recombinant plasmid pETT668, which was then transformed into E. coli The expression strain DE3 competent cells, induced by IPTG, expressed by SDS PAGE. Results The molecular mass of pETT668 protein was 49ku. The protein expression of pETT668 increased gradually with the induction time, and peaked at 5h after induction. The results of TLC showed that the target protein accounted for 34.6% of the total bacterial protein. Western blotting showed that the expressed protein was recognized by the serum of infected Trichinella domestic pigs. Conclusions The larval stage specific T668 gene of Trichinella spiralis is highly expressed in Escherichia coli, and the expressed protein has good antigenicity.