鸡柔嫩艾美尔球虫HAP2基因的克隆与原核表达

Cloning and Prokaryotic Expression of Eimeria.tenella HAP2 Gene

为研究鸡柔嫩艾美尔球虫(E.tenella)HAP2(hapless2/generative cell-specific)蛋白的抗原性,根据Gen?Bank发表的HAP2基因的cDNA序列(Gene ID:25252561)ORF设计1对引物,用RT-PCR方法从柔嫩艾美耳球虫配子体总RNA中扩增HAP2基因,将扩增产物与pMD18-T载体连接后进行测序和序列分析,将测序正确的HAP2基因亚克隆入原核表达载体pET32a,转化宿主菌E.coli RGB,在IPTG诱导下进行原核表达。结果在20℃诱导12 h条件下成功表达了HAP2融合蛋白大小为41 ku,并应用磁珠纯化的方法纯化了重组蛋白,获得了HAP2融合蛋白,为进一步研究HAP2的抗原性奠定基础。

In order to study the antigenicity of E.tenella HAP2 gene in chicken, a pair of primers was designed based on the cDNA sequence of the HAP2 gene published by GeneBank(Gene ID:25252561) ORF. The HAP2 gene was amplified by RT-PCR from the total RNA of the gametophyte of E.tenella. The amplification product was linked to the pMD18-T for sequencing and sequence analysis. The correct sequencing of the HAP2 gene was cloned into the prokaryotic expression vector pET32 a, and converted to host bacterium E. coli RGB. Fusion protein was induced by IPTG and successfully expressed under the condition of 20 hours at 20 ℃. The fusion protein of HAP2 is 41 kDa and was purified by magnetic bead purification method, which laid the foundation for further research on the antigenicity of HAP2.