细粒棘球绦虫EgG1Y162抗原基因的序列分析及其蛋白表达

发布时间：2018-08-21 08:09

【摘要】： 目的:克隆egG1Y162抗原基因,构建PET-41a/egG1Y162原核表达质粒,并诱导表达egG1Y162重组蛋白及抗原性检测。方法:根据emY162基因序列设计引物,分别从细粒棘球绦虫原头蚴和成虫两个发育阶段,提取基因组DNA和总RNA,mRNA反转录为cDNA,利用PCR的方法以基因组DNA和cDNA为模板扩增egG1Y162基因;构建PUCm-T/egG1Y162重组质粒,经PCR、酶切及测序鉴定后,测序确定其正确性。利用定向克隆技术将egG1Y162抗原基因片段克隆至原核表达质粒PET-41a上,通过酶切分析和PCR鉴定筛选出阳性克隆,测序确定序列。IPTG初步诱导和表达egG1Y162-GST重组蛋白,通过SDS-PAGE电泳和Western blot试验分析鉴定。结果:从细粒棘球绦虫的两个不同发育阶段均克隆出egG1Y162基因,从总DNA克隆得到片段长度1 680bp,从cDNA克隆得到片段长度459bp。相似性比较表明,egG1Y162基因序列与emY162相似性为91%,而egG1Y162基因cDNA与emY162相似性为95%。进一步分析显示,egG1Y162基因序列由3个外显子和2个内含子组成,外显子区域分别为1～70,1 064～1 380和1 577～1 648。位于疏水端1～16位氨基酸构成egG1Y162信号肽序列,35～115位氨基酸形成一个大的纤黏连蛋白剪接体FN3,133～152位氨基酸构成羧基端跨膜区域。测序结果显示egG1Y162抗原基因长度为360 bp,编码120个氨基酸。构建的PET-41a/egG1Y162原核表达质粒,经IPTG诱导后,SDS-PAGE检测表明egG1Y162-GST重组蛋白得到成功表达,在相对分子量为44 KDa处有表达条带。Western blot分析显示阳性印迹条带,分子量为44 KDa,egG1Y162重组蛋白能与细粒棘球蚴感染40天后犬的血清发生反应;与包虫病患者血清也有阳性反应。结论:成功克隆egG1Y162抗原基因,序列对比分析显示egG1Y162的cDNA与emY162的cDNA具有很高的相似性,基因的差异性主要存在于内含子区域,egG1Y162抗原基因是一种新的基因。成功诱导表达出egG1Y162重组蛋白,并且egG1Y162重组蛋白具有很好的抗原性。
[Abstract]:Objective: To clone egG1Y162 antigen gene, construct PET-41a/egG1Y162 prokaryotic expression plasmid, induce the expression of egG1Y162 recombinant protein and detect its antigenicity. Methods: According to the sequence of emY162 gene, primers were designed to extract genomic DNA and total RNA from protozoan and adult stages of Echinococcus granulosus, and mRNA was retranscribed into cDNA. Methods The egG1Y162 gene was amplified with genomic DNA and cDNA as templates, and the PUCm-T/egG1Y162 recombinant plasmid was constructed and identified by PCR, restriction enzyme digestion and sequencing. The recombinant protein of egG1Y162-GST was induced and expressed by IPTG, and identified by SDS-PAGE electrophoresis and Western blot. Results: The egG1Y162 gene was cloned from two different developmental stages of Echinococcus granulosus, and the length of the fragment was 1 680 BP from the total DNA clone. The length of the fragment was 459 BP from the cDNA clone. The sequence similarity of egG1Y162 gene to emY162 was 91%, while that of egG1Y162 gene was 95%. The sequence analysis showed that the length of egG1Y162 antigen gene was 360 bp, encoding 120 amino acids. The constructed prokaryotic expression plasmid of ET-41a/egG1Y162 was induced by IPTG. SDS-PAGE analysis showed that the recombinant protein of egG1Y162-GST was obtained. Western blot analysis showed that the recombinant protein could react with the serum of dogs 40 days after infection with Echinococcus granulosus, and positive reaction with the serum of patients with hydatidosis. The analysis showed that the cDNA of egG1Y162 was highly similar to that of emY162. The gene difference mainly existed in the intron region. The egG1Y162 antigen gene was a new gene. The recombinant protein of egG1Y162 was successfully induced and expressed, and the recombinant protein of egG1Y162 had good antigenicity.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】

相关期刊论文 前10条

1 陈根,史大中;包虫病治疗的现状概况[J];地方病通报;2001年04期

2 王俨,林仁勇,丁剑冰,卢晓梅,张静萍,王星,梁晓慧,张亚楼,张琰,温浩;泡球蚴18基因的克隆、原核表达质粒的构建及其初步表达的研究[J];新疆医科大学学报;2005年01期

3 石佑恩,韩家俊,李文桂,石星,李传明,邓伟文,皇甫永穆,郑波,程继忠,冯作化,梁驹卿,肖红,路艳艳,曾星,海涛;日本血吸虫基因工程BCG多价疫苗的研究[J];中国血吸虫病防治杂志;1998年S1期

4 郑宏,徐志新;宿主感染细粒棘球蚴免疫反应的研究进展[J];中国寄生虫病防治杂志;2002年01期

5 朱佑明,李文桂;细粒棘球绦虫分子生物学研究进展[J];中国寄生虫病防治杂志;2005年03期

6 曹春宝;丁剑冰;马秀敏;;棘球蚴感染中的免疫逃避相关分子[J];中国病原生物学杂志;2008年10期

7 沈蕾,张兆松;CD4~+T细胞与血吸虫感染的保护性免疫[J];中国寄生虫学与寄生虫病杂志;2001年04期

8 余森海;;棘球蚴病防治研究的国际现状和对我们的启示[J];中国寄生虫学与寄生虫病杂志;2008年04期

9 韩秀敏;王虎;邱加闽;马宵;蔡辉霞;刘培运;丁启军;代南;Ito.A;Craig PS;;青海省班玛县泡型和囊型包虫病流行现状调查分析[J];中国人兽共患病学报;2006年02期

10 丁剑冰;马秀敏;魏晓丽;林仁勇;王俨;张静萍;温浩;;细粒棘球绦虫Eg95基因疫苗和重组抗原诱导小鼠免疫应答的比较研究[J];中国人兽共患病学报;2006年04期

，

本文编号：2195090