柔嫩艾美耳球虫配子体基因Etgam56的克隆表达与鉴定

Cloning, expression and characterization of the Etgam56 gene of Eimeria tenella

提取柔嫩艾美耳球虫(扬州株)配子体基因组DNA,PCR扩增配子体基因Etgam56,经克隆和序列分析后,优化密码子,连接至pET-28a(+),构建原核表达载体pET-28a(+)-Etgam56,优化条件进行体外诱导表达,Western blot分析其抗原性。结果显示,Etgam56基因全长1425 bp,为一个完整开放阅读框,共编码474个氨基酸;体外表达重组蛋白的大小约为56 ku,IPTG终浓度为0.10 mmol/L,37℃诱导3 h时的表达量最高,且主要以包涵体形式存在。Western blot结果显示,该重组蛋白能被6×HIS标签单克隆抗体、柔嫩艾美耳球虫鸡康复血清和毒害艾美耳球虫鸡康复血清识别,表明该重组蛋白为特异性表达的蛋白,具有很好的抗原性和交叉反应性。本研究将为进一步探析柔嫩艾美耳球虫配子体蛋白的功能与研制鸡球虫基因工程疫苗奠定基础。

In order to investigate the function of the gam56 gene of Eimeria tenella,genomic DNA was extracted from the oocysts of E.tenella(the Yangzhou strain),and was used as a template to amplify the gam56 gene encoding the gametocyte protein.After cloning,sequencing analysis and codon optimization,the Etgam56 gene was subcloned to a pET-28 a(+)vector to construct a prokaryotic expression vector pET-28 a(+)-Etgam56.After being transformed into Escherichia coli BL21,the recombinant plasmid was induced to express by IPTG.To identify the optimal inducing conditions,the main factors of expression conditions,including IPTG concentration,induction time,and temperature,were examined by SDS-PAGE.The results showed that the Etgam56 gene was composed of 1425 bp,coding 474 amino acids.The recombinant protein had a molecular weight of 56 ku,and had maximum expression at 37℃for 3 hours by 0.10 mM IPTG.Western-blot analysis indicated that the recombinant protein was recognized specifically by anti-6×HIS monoclonal antibodies and the recovery serum from chickens infected with E.necatrix or E.tenella sporulated oocysts,respectively.This study laid a foundation for further research on characterization of Etgam56 and development of recombinant subunit vaccines against coccidiosis.