旋毛虫新生幼虫期特异性T668 cDNA在大肠埃希菌中的表达及鉴定

EXPRESSION AND IDENTIFICATION OF NEWBORN LARVAE STAGE-SPECIFIC GENE T668 cDNA OF TRICHINELLA SPIRALIS IN ESCHERICHIA COLI

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 newborn larvae stage-specific T668 cDNA of Trichinella spiralis in Escherichia coli, so as to obtain genic engineer antigen. Methods T668 cDNA with no signal peptide sequence was obtained from pBK-CMV-T668 by PCR, and subcloned into prokaryotic expression vector pET28a (+), and the recombinant expression plasmid (pETT668) was constructed. Then, the expression plasmid was transformed into E. coli strain DE_~3 and induced by IPTG, the expressed protein was identified by SDS-PAGE. Results The molecular weight of the expressed protein was 49 ku, the quantity of the expressed protein increased with time extending and reached 34.6% of the total bacterial protein after inducing 5 hours. Western blotting showed the expressed protein was recognized by positive sera from swine infected with T. spiralis. Conclusion Newborn larvae stage-specific T668 cDNA of T. spiralis was expressed and identified, and the expressed protein had satisfactory antigenicity.