旋毛虫新生幼虫编码p46 kDa抗原基因的克

发布时间：2018-01-27 04:10

本文关键词： 旋毛虫新生幼虫 cDNA 文库 p46 kDa 抗原 WN10 cDNA 克隆表达抗原性　出处：《吉林大学》2005年硕士论文　论文类型：学位论文

【摘要】：旋毛虫病是由旋毛虫(Trichinella spiralis)引起的一种呈全球性分布的人兽共患寄生虫病,严重威胁着人民群众的身体健康。本研究利用分子生物学及免疫学技术,分离新生幼虫抗原基因,从而为旋毛虫病诊断抗原及保护性抗原的筛选及大量制备奠定基础。以旋毛虫感染猪血清为抗体探针,对旋毛虫新生幼虫cDNA文库进行免疫筛选,从4×105个重组噬菌体中共获得156个阳性噬菌斑。其中WN10 cDNA 编码旋毛虫新生幼虫p46 kDa抗原,全长为1352 bp,含有1218bp完整的开放阅读框,氨基酸序列第1-18位为信号肽。GenBank数据库BlastN 同源性分析表明,与旋毛虫Ts ORF9.10 (U88241) 同源性为99%(899/906)。研究表明此基因在旋毛虫肌幼虫、新生幼虫、3日龄及5 日龄成虫期均有表达。利用PCR 技术, 从筛选得到的pBK-CMV-WN10重组质粒中扩增到不含信号肽序列的WN10基因片段,重组到原核表达载体pET-28a中。用IPTG诱导培养重组表达菌,对表达产物进行SDS-PAGE分析,结果显示,经IPTG诱导后重组菌体裂解产物与对照菌相比出现了1条相对分子量约为48 kDa的新条带。重组蛋白的抗原性分析表明,该重组抗原对小鼠产生了较好的免疫保护效果,其旋毛虫肠道成虫和肌幼虫的减虫率分别为64.28%和61.21%,说明重组抗原可诱导小鼠机体获得较强的抗旋毛虫攻击感染的能力及产生抗旋毛虫在肌肉组织中寄生的免疫保护作用。Western-blotting 检测显示,重组抗原可被旋毛虫感染的猪血清、小鼠血清以及重组抗原免疫的小鼠血清识别,与ELISA 检测结果一致,说明在旋毛虫感染动物及重组抗原免疫后,可以刺激宿主免疫系统使其产生抗此抗原的特异性抗体应答,重组抗原具有很好的抗原性,从而为旋毛虫病诊断抗原及保护性抗原的筛选及大量制备奠定基础。
[Abstract]:Trichinella spiralis is a globally distributed zoonotic parasitic disease caused by Trichinella spiralis. It is a serious threat to the health of the people. In this study, the antigen genes of newborn larvae were isolated by molecular biology and immunology. Therefore, it will lay a foundation for the screening and preparation of diagnostic and protective antigens of trichinellosis. The cDNA library of newborn larvae of Trichinella spiralis was screened by using sera from pigs infected with Trichinella spiralis as antibody probe. A total of 156 positive plaque were obtained from 4 脳 105 recombinant phages, in which WN10 cDNA encoded p46 kDa antigen from newborn larvae of Trichinella spiralis, with a length of 1352bp. It contains 1218bp complete open reading frame, and the amino acid sequence 1-18 is the signal peptide. The BlastN homology analysis of GenBank database shows that. The homology of this gene with ts ORF9.10 U88241 was 9999 / 906. The results showed that the gene was found in muscle larvae and newborn larvae of Trichinella spiralis. The WN10 gene fragment without signal peptide sequence was amplified from the pBK-CMV-WN10 recombinant plasmid by PCR technique. Recombinant into prokaryotic expression vector pET-28a. The recombinant expression bacteria were induced by IPTG. The expression product was analyzed by SDS-PAGE. After induction by IPTG, a new band with a relative molecular weight of about 48 kDa was found in the recombinant cell lysis product compared with the control strain. The antigenicity analysis of the recombinant protein showed that the recombinant antigen had a good immune protection effect on mice, and the worm reduction rates of adult and muscle larvae of Trichinella spiralis were 64.28% and 61.21%, respectively. These results indicate that recombinant antigen can induce mice to acquire stronger ability to resist Trichinella spiralis attack infection and to produce anti-Trichinella spiralis parasitic immunity protection in muscle tissue. Western-blotting. The test showed. The recombinant antigen can be recognized by the pig serum infected by Trichinella spiralis, mouse serum and mouse serum immunized with recombinant antigen, which is consistent with the results of ELISA detection, indicating that after Trichinella spiralis infection and recombinant antigen immunization. It can stimulate the host immune system to produce a specific antibody response against this antigen, and the recombinant antigen has good antigenicity, thus laying a foundation for screening and preparing a large number of diagnostic and protective antigens for trichinellosis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392;Q78

【相似文献】