摘要
通过设计、合成引物,以旋毛虫RNA为模板,用RT-PCR法扩增出旋毛虫P49序列,并进行了序列测定。将P49基因亚克隆至表达载体pET-28b中,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,作SDS-PAGE及Western blot分析。结果表明,PCR法扩增出P49序列,其大小约为960bp,将构建的重组质粒pGEM-P49进行序列测定表明其与Genbank中的P49序列具有高度的同源性。成功构建了重组表达载体pET-P49;SDS-PAGR及Western blot分析表明,表达产物分子量约为38kDa,约占菌体总蛋白的7％左右,且能被感染旋毛虫猪阳性血清所识别。
Primers were designed and synthesized. Using worm RNA as a template, the DNA encoding excretory-secretory products of Trichinella spiralis was amplified by reverse transcriptase-polymerase chain rection(RT-PCR). The gene encoding P49 was cloned and sequenced. To subcolone the P49 gene into vector SK( + ) and then into vector pET-28b, and transform the recombinant plasmid into competent cells of E.coli BLzi. The expressed protein induced by IPTG was analyzed with SDS-PAGE and Western blot. The results showed that a specific band about 0. 96Kb was amplified by RT-PCR. There was highly homology of fragment to be sequenced with that of P49 gene in Genbank. Molecular weight of expressed protein was about 38kDa and content, approximate 7% of whole bacterial lysate. The expressed product could be recognized by positive sera from swine infected with Trichinella spiralis.
出处
《中国兽医杂志》
CAS
北大核心
2003年第7期7-10,共4页
Chinese Journal of Veterinary Medicine
基金
湖北省畜牧局2001年重点项目
