鸡堆型艾美耳球虫gam56基因的克隆及其表达产物的免疫原性分析

Cloning of gam56 gene from Eimeria acervulina and immunogenicity analysis of its expressed product

为了研究堆型艾美耳球虫gam56基因的功能,提取堆型艾美耳球虫(扬州株)卵囊DNA,应用PCR扩增gam56基因,克隆至pGEM-T-Easy载体;测序、密码子优化后连接至pET-28a质粒,构建pET-28aEagam56表达载体,转化至大肠杆菌BL21;重组菌经酶切和测序鉴定后诱导表达,对表达产物进行SDS-PAGE分析和Western-blot鉴定。结果显示:gam56基因全长1 323 bp,编码440个氨基酸;重组蛋白大小约49 ku,以可溶蛋白形式为多,能被组氨酸单抗特异性识别;该重组蛋白能被小鼠抗重组蛋白多克隆抗体以及鸡抗堆型艾美耳球虫、鸡抗毒害艾美耳球虫、鸡抗巨型艾美耳球虫和鸡抗柔嫩艾美耳球虫的阳性血清特异性识别。本研究结果为进一步探析堆型艾美耳球虫gam56基因功能与研制鸡球虫病基因工程疫苗奠定了基础。

In order to investigate the function of gam56 gene of Eimeria acervulina,DNA was extracted from oocysts of E.acervulina(Yangzhou strain),and was used as a template for PCR to amplify the gam56 gene encoding gametocyte protein. The gam56 gene was inserted to p GEM-T-Easy vector by TA cloning. After sequencing analysis and codon optimization,the gam56 gene was subcloned to p ET-28 a vector to obtain a recombinant plasmid pET-28 a-Eagam56. After being transformed into Escherichia coli BL21,the recombinant plasmid was induced to express by IPTG,which was vertified by SDS-PAGE and Western-blot analysis. The results showed that the gam56 gene was composed of 1 323 bp,coding 440 amino acids. The recombinant protein had a molecular weight of 49 ku and was predominately expressed in the solubility. Western-blot analysis indicated that the recombinant protein could be recognized specifically by anti-His monoclonal antibodies,anti-recombinant protein polyclonal antibodies from mice and the serum of chicken infected with E.acervulina,E.necatrix,E.maxima or E.tenella,respectively.These findings lays a foundation for further study on function of gam56 gene of E.acervulina and development of recombinant subunit vaccines against coccidiosis.