柔嫩艾美耳球虫表面抗原SAG2基因的克隆与表达

根据生物信息学预测的基因序列设计引物,应用RT-PCR方法从柔嫩艾美耳球虫第二代裂殖子总RNA扩增获得了鸡柔嫩艾美耳球虫表面抗原2(surface antigen 2,SAG2)基因序列,将其与pGEM-T easy载体连接后转化E.coliDH5α,筛选阳性克隆,以带有限制酶切位点的特异性引物用PCR方法扩增不含SAG2 N端信号肽序列的ORF序列后克隆至表达载体pET-32 a(+),构建了重组表达质粒pET-32 a(+)-SAG2,并将其转化至E.coliBL21(DE3)。经IPTG诱导,获得了SAG2重组抗原在大肠杆菌的高效表达,重组蛋白的表达量约占菌体总蛋白的35%,融合蛋白的分子量约为47 ku。菌体经超声处理后进行SDS-PAGE分析表明,表达蛋白以包涵体的形式存在。

Cloning and expression of the gene encoding SAG2 of Eimeria tenella

The gene encoding the surface antigen 2 of E.tenella(EtSAG2) Yangling strain was amplified by RT-PCR.The PCR product was purified and cloned into pGEM-T easy vector and the recombinant plasmid was sequenced.The EtSAG2 ORF without the signal sequence was amplified by PCR from the pGEM-SAG2 using the specific primers and cloned into the expression vector pET-32a( ),and then was transformed into E.coli BL 21(DE3).After conformed by PCR with endonuclease digestion,the cloned gene was sequenced and aligned(online) with BLAST program.The result showed the cloned sequence of EtSAG2 from E.tenella Yangling strain was highly similar to that of(E.tenella) H strain with an identity of 99.75%.The gene EtSAG2 without signal sequence in recombinant plamid pET-32a( )-EtSAG2 could be expressed in inclusion body of E.coli BL 21 when induced by IPTG,and the molecular size of recombinant protein was 47 ku(revealed) by SDS-PAGE.