【摘要】 为通过E.coli表达系统制备猪细小病毒NS1抗原表位区蛋白,本试验利用DNAstar软件确定含有抗原表位的NS1蛋白片段,然后运用PCR方法从含有NS1基因的载体中扩增NS1抗原表位区基因片段,并将该基因片段插入到原核表达载体pGEX-4T-1中,构建成与GST标签融合的NS1抗原表位基因的表达载体pGEX-GST-NS1,将表达载体转化BL21大肠杆菌筛选出工程菌。经IPTG诱导表达NS1融合蛋白,经GST-琼脂糖凝胶磁珠分离纯化,制备出与GST融合的NS1抗原表位蛋白。经SDS-PAGE以及Western blot鉴定,制备的NS1融合蛋白分子量和预期一致,并且可以和抗GST标签抗体进行特异反应,表明NS1抗原蛋白已成功制备,为下一步NS1抗体的研制提供了必要的条件。更多还原

【Abstract】 To prepare porcine parvovirus NS1 protein fragment containing epitopes using E.coli,NS1 epitope fragment was first deter mined using DNAstar software and amplified by PCR from the vector containing full-length NS1 gene.The NS1 epitope fragment then was inserted into pGEX-4 T-1 plasmid to construct the prokaryotic expression vector of GST tagfused NS1 antigen gene,pGEX-GST-NS1.The expression vector was transformed into BL21 E.coli to generate NS1 engineering E.coli strain,which was cultured under the induction of IPTG for expression of NS1 epitope protein.The expressed GST-fused NS1 epitope protein was isolated and purified by GST-magnetic agarose beads.The prepared protein was identified by SDS-PAGE and Western blot to have the same molecular weight as expected and can specifically bind to the anti-GST antibody,indicating that the NS1 antigen protein is successfully prepared.This study provides the necessary conditions for future preparation of NS1 antibody.更多还原