猪传染性胃肠炎病毒S基因A抗原位点序列的克隆与原核表达

将RT-PCR扩增的猪传染性胃肠炎病毒(TGEV)S基因A抗原位点目的片段克隆、双酶切后连接于表达载体,经酶切、PCR和测序鉴定的阳性重组质粒转化BL21(DE3),用IPTG诱导,进行SDS-PAGE和Western-blotting分析,确定目的蛋白的原核表达情况和免疫特异性。结果显示,目的片段的大小为534bp,序列分析表明,该基因与其他TGEV相应基因具有很高的同源性;Western-blotting检测可见分子质量约43 ku的融合蛋白条带,表明,该蛋白具有良好的反应原性。

Cloning and prokaryotic expression of antigenic site A sequence of S gene of swine transmissible gastroenteritis virus

Antigenic site A sequence of S gene of swine transmissible gastroenteritis virus(TGEV) was amplified by RT-PCR and cloned into the prokaryotic expression vector after digestion with restriction enzyme.The positive recombinants identified by enzymatic digestion,PCR and sequencing was transformed into Escherichia coli BL21 and the recombinant transformant was induced with IPTG.The prokaryotic expression and the immunologic specificity of the expressed protein were analyzed by SDS-PAGE and Western-blotting.The results showed that the target gene was 534 bp in length and had a relatively high homology with the corresponding gene from other TGEV strains.The expressed fusion protein was 43 ku in size,and had good reactogenicity as revealed by Western-blotting.