猪传染性胃肠炎病毒S基因抗原位点B与C之间片段的克隆与原核表达

Cloning and prokaryotic expression of the fragment between antigenic sites B and C of S gene of transmissible gastroenteritis virus

将RT-PCR扩增的猪传染性胃肠炎病毒S基因抗原位点B与C之间的目的片段经克隆、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诱导重组菌株表达其融合蛋白。结果显示,目的片段的大小为357bp,序列分析表明,S基因与其他猪传染性胃肠炎病毒相应基因具有很高的同源性;Western-blotting检测表明,分子质量约34ku的融合蛋白具有良好的反应原性。

The fragment between antigenic sites B and C of S gene of swine transmissible gastroenteritis virus（TGEV） was amplified by RT-PCR and cloned into the prokaryotic expression vector after digestion. The positive recombinant plasmid,which was identified by enzymatic digestion, PCR and sequencing, was transformed into BL21 strain and the recombinant transformants were 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 fragment was 357 bp in length and it had a relatively high homology with the corresponding fragment from other TGEV strains in nucleotide and deduced amino acid sequences. Western-blotting analysis showed that the fusion protein of 34 ku in molecular mass had good reactogenicity.