PRRSV ORF5和ETEC faeG基因的克隆及在毕赤酵母中的表达

Cloning and expression of porcine reproductive and respiratory syndrome virus ORF5 gene and enterotoxigenic Escherichia coli faeG gene in Pichia pastoris

分别以猪繁殖与呼吸综合征病毒(PRRSV)分离株JL-12的cDNA和产肠毒素大肠杆菌质粒为模板,PCR扩增ORF5和faeG基因,对扩增产物进行鉴定和测序,并构建重组表达质粒pGAPZαA-ORF5、pGAPZαA-ORF5-FaeG,将pGAPZαA-ORF5与pGAPZαA-ORF5-FaeG分别转入毕赤酵母表达系统中构建重组菌,获得分泌表达蛋白的重组酵母菌,对其表达产物进行SDS-PAGE和Western-blot分析。SDSPAGE分析结果显示,分别得到分子质量约为24ku的GP5蛋白和52ku的GP5-FaeG融合蛋白。Western-blot分析结果显示,GP5蛋白、GP5-FaeG融合蛋白都具有反应原性。上述研究结果为PRRSV的分子生物学功能和基因工程疫苗的研发奠定了基础。

Using the cDNA of porcine reproductive and respiratory syndrome virus（PRRSV）,which isolated from Jilin named as JL-12,and enterotoxigenic Escherichia coli（ETEC）plasmid as template,ORF5 and faeGgenes were amplified by PCR and sequenced.The constructed recombinant plasmids pGAPZαAORF5and pGAPZαA-ORF5-FaeG were respectively transformed into Pichia pastoris expression system to construct recombinant bacteria and acquire recombinant yeast which secretes and expresses the proteins.The expressed products were verified by SDS-PAGE and Western-blot.In result,GP5 protein and GP5-FaeG fusion protein were acquired with good reactionogenicities,and GP5 protein and GP5-FaeG fusion protein were approximately 24 ku and 52 ku in molecular mass,respectively.The above-mentioned result laid the foundations for the molecular biological function and development of genetic engineering vaccine of PRRSV.