摘要
筛选旋毛虫肠道1期幼虫抗原性基因。利用λZAP载体构建旋毛虫肠道1期幼虫cDNA文库。用感染旋毛虫26 d猪血清对其进行免疫学筛选,对筛选出的阳性克隆进行测序与分析;应用RT-PCR方法对编码p46 000蛋白强阳性克隆基因在不同发育时期虫体转录情况进行检测。成功构建了旋毛虫肠道1期幼虫cDNA文库,其库容量为1.5×106pfu,重组率为95%,插入片段在400 bp^2 000 bp之间,扩增后文库滴度为1.5×1012pfu/mL。筛选出阳性克隆33个,序列分析表明,有20个克隆为同一已知基因,称为旋毛虫p46 000蛋白,编码旋毛虫半胱氨酸蛋白酶抑制剂,且在筛选过程中反应信号均较强;还发现了一些旋毛虫新基因,如编码Ubc蛋白,GM13181蛋白,Rieske硫铁蛋白1等。本研究获得了一个高丰度、反应原性较强抗原基因,其编码蛋白为半胱氨酸蛋白酶抑制剂。
To obtain antigenic genes from Trichinella spiralis L1 at intestinal stage, the cDNA library of L1 at intestinal stage from T. spiralis was successfully constructed with the lambda ZAP vector and immunoscreened by using 26 days anti-serum of the pig infected with T. spiralis, and the obtained positive clones were sequenced and analyzed. The transcriptions of p46 000 gene in the different stages of Trichinella larvae were displayed by RT-PCR. The size of the primary library was 1.5 × 10^6 with 95% recombinant rate and the titer of the amplified library was 1.2 × 10^12 pfu/mL. 33 positive clones were obtained by immuno-screening,and 20 clones of them encoded the same known gene EU263325, called p46 000 protein and encodes a novel cystatin-like protein. They showed stronger reactive signal during immuno-screening. Meanwhile, some new genes found, for example, Uhc protein,GM13181 protein,Rieske iron-sulfur protein 1 and so on. The antigenic gene, which encodes hypothetical protein, with strong reactionogenicity and high copy was obtained.
出处
《动物医学进展》
CSCD
北大核心
2009年第8期50-54,共5页
Progress In Veterinary Medicine
基金
国家杰出青年基金(30825033)
国家863专题项目(2006AA02Z451)
关键词
旋毛虫
肠道1期幼虫
抗原基因
筛选
Trichinella spiralis
L1 at intestinal stage
antigenic genes
screening
