旋毛虫肌幼虫p45cDNA的克隆及鉴定  

Cloning and identification of the p45 cDNA from Trichinella spiralis

作　　者：孙树民[1] 吴秀萍[1] 付宝权[1] 王学林[1] 郭恒[1] 于申业[1] 邓洪宽[1] 刘马峰[1] P.Boireau 刘明远[1] 

机构地区：[1]吉林大学人兽共患病教育部重点实验室人兽共患病研究所

出　　处：《中国人兽共患病学报》2008年第5期439-441,445,共4页Chinese Journal of Zoonoses

基　　金：国家863计划项目(2006AA02Z451);国家自然科学基金(30771885);国际科学基金(B/352521)

摘　　要：目的本研究根据GenBank中旋毛虫45kDa抗原基因的序列设计引物,采用PCR技术从旋毛虫肌幼虫cDNA文库中扩增靶基因p45cDNA,并克隆到pMD-18T载体,转化至大肠埃希氏菌NovaBlue后测序分析。获得827bp的p45cD-NA。该序列与GenBank中的旋毛虫p45基因序列相比共有1个核苷酸发生改变,二者的同源性为99%,其编码蛋白质由268个氨基酸组成,与45kDa抗原的同源性为99%。将p45cDNA克隆到原核表达载体pET28a并转化至表达菌BL21star(DE3)后,经IPTG诱导表达出约30kDa的重组蛋白。Western-blot检测表明,p45重组蛋白可以被旋毛虫感染猪血清识别,具有良好的抗原性。According to the sequence of the 45 kDa antigen gene of Trichinella spiralis published in GenBank,the specific primers were designed,and were used to amplify the target gene p45 cDNA of T. spiralis muscle larvae by PCR. The amplified product was cloned into vector pMD-18T and then transformed to E. coli NovaBlue and sequenced. In this way, the P45 cDNA with 827 bps was thus obtained. Compared with the p45 gene sequence in GenBank, only one nucleotide difference could be detected,approaching up to a 99 % homology. The deduced protein composed of 268 amino acids,also with a 99% homology with the 45 kDa protein. When p45 cDNA was cloned into prokaryotic expression vector pET28a and then transformed to E. coli BL21（DE3）,a 30 kDa recombinant protein was expressed after IPTG induction. As demonstrated by Western blot assay, the recombinant protein could be recognized by pig T. spiralis antiserum, indicating the presence of antigenicity of this protein.

关 键 词：旋毛虫 P45 CDNA 克隆 表达 抗原性 

分 类 号：S852.7[农业科学—基础兽医学] Q78[农业科学—兽医学]