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目的 获得旋毛虫排泄分泌抗原(excretory antigen,ES)p49基因的克隆、测序及表达。 方法 通过RT-PCR,从旋毛虫幼虫总RNA中扩增得到特异片段,利用TA克隆将PCR产物克隆入pUC-T载体中并进行测序及同源性比较,并定向克隆到表达载体pEG-4T-3中,转化感受态细胞,诱导表达。 结果 RT-PCR扩增得到p49基因,其核苷酸序列与已发表的p49基因序列一致;BLAST分析表明其与旋毛虫p49基因、43 KDa分泌性糖蛋白同源性均为99％。经SDS-聚丙烯酰胺凝胶电泳,重组蛋白诱导表达在67 KDa处有一新蛋白带。 结论 提取旋毛虫幼虫总RNA,用RT-PCR方法克隆并表达了p49基因。
Objective To obtain the cloning, sequencing and expression of p49 gene of Trichinella spiralis excreteory antigen (ES). Methods The specific fragment was amplified by RT-PCR from the total RNA of Trichinella spiralis larvae. The PCR products were cloned into pUC-T vector by TA cloning and sequenced and compared by homology, and cloned into the expression vector pEG-4T -3, transformed into competent cells to induce expression. Results The p49 gene was amplified by RT-PCR and its nucleotide sequence was consistent with the published sequence of p49 gene. BLAST analysis showed that it shared 99% homology with Trichinella p49 gene and 43 KDa secreted glycoprotein. After SDS-polyacrylamide gel electrophoresis, the recombinant protein induced expression of a new protein band at 67 KDa. Conclusion The total RNA of Trichinella spiralis larvae was extracted and the p49 gene was cloned and expressed by RT-PCR.