巨型艾美球虫配子体gam82基因的克隆表达与免疫原性分析  

Cloning and prokaryotic expression of gam82 of Eimeria maxima NT strain and immunogenicity analysis of the recombinant protein

作　　者：彭昊[1] 陶建平[1] 许金俊[1] 刘丹丹[1] 李建梅[1] 任春芝[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《中国兽医学报》2010年第9期1203-1207,1212,共6页Chinese Journal of Veterinary Science

基　　金：江苏省自然科学基金资助项目(B2007079)

摘　　要：提取巨型艾美球虫(Eimeria maxima)南通株(NT株)配子体总RNA,应用RT-PCR技术对gam82基因进行克隆,并对其核苷酸及氨基酸进行序列分析。结果表明,克隆的基因全长为1791个核苷酸,具有一个完整的开放阅读框,编码596个氨基酸,与GenBank中发表的Houghton株gam82基因同源性为100%。根据对推导的氨基酸序列的二级结构和抗原表位分析的结果,筛选免疫原性良好的区域,命名为gam82-Y,进行原核表达。重组菌经过IPTG诱导后,通过SDS-PAGE电泳和Western-blot检测,gam82-Y基因片段在大肠杆菌中得到了融合表达,相对分子质量约为53000。可溶性分析表明,融合蛋白主要以包涵体形式存在。以E.maxima高免血清为一抗进行Western-blot检测,显示重组抗原gam82-Y具有免疫原性。Gam82gene of Eimeria maxima NT strain isolated from Nantong city was amplified from total RNA of purified gametocytes by reverse transcription polymerase chain reaction （RT-PCR）.The results indicated that gam82gene of E.maxima NT strain included an opening read frame consisted of 1 791bp which encoded a polypeptide of 596amino acids.Homology of the ORF sequences of gam82 between E.maxima NT strains and E.maxima Houghton strains was 100%.The antigenic index,secondary structure of gam82 protein of NT strain were predicted by using various methods and computer software to select the appropriate peptide sequence.The gam82gene fragment of E.maxima NT strain,which was named gam82-Y,was amplified by polymerase chain reaction（PCR）from plasmid pGEM-T-gam82,and subcloned into express vector pGEX-6P-1.After induced by IPTG,the 53 000 GST- gam82-Y fusion protein was expressed,which were confirmed by SDS-PAGE and Western-blot analysis,respectively.The fusion protein was expressed as insoluble form,and showed to be immunogenic according to the result of Western-blot analysis using the positive serum of chicken infected with E.maxima.

关 键 词：巨型艾美球虫 gam82基因 克隆 序列分析 表达 

分 类 号：S852.729[农业科学—基础兽医学]