摘要
人工合成VP73基因全长序列,利用DNAstar软件确定其中抗原性较高的区域,利用Primer Premier5.0设计一对特异性引物,通过PCR扩增得到243bp的非洲猪瘟病毒VP73基因片段(vp73l)。将该片段与表达载体pET-32a连接克隆至大肠杆菌Escherichia coli(E.coli)DH5α菌株,经测序、菌落PCR和酶切鉴定,选取VP73L基因正向插入,读码框正确的阳性克隆。构建重组质粒并转化BL21(DE3),经IPTG诱导,融合蛋白以可溶性形式在E.coliBL21(DE3)高效表达,经His亲和层析柱得以纯化。Western blotting显示,该融合蛋白能与兔抗非洲猪瘟病毒VP73多克隆抗体发生特异性反应。所得结果为进一步研究:分离纯化该重组融合蛋白;确证目标抗原蛋白VP73L免疫原性及其特异性,进而达到建立非洲猪瘟病毒的免疫检测方法奠定了必要的材料与技术基础。
A full-length VP73 gene sequence was synthesized,the its higher antigen area is predicted by using DNAStar software,and one pair of specific primers for this area are designed by using Primer Premier 5.0.Amplified 243bp fragment of the VP73 gene（vp73l） by PCR was digested and cloned into the prokaryotic expression vector pET-32a.The positive clone of inserting VP73L gene with correct reading frame was confirmed by sequencing and colony PCR.After induction by IPTG,the fusion protein was highly expressed in Escherichia coli BL21（DE3） in soluble form.The recombinant protein was purified with His-Bind affinity chromatography.Western blotting analysis revealed that the recombinant protein could react with rabbit anti-African swine fever virus VP73 polyclonal antibody.Fusion expression of African swine fever virus VP73L is helpful to to prepare ASFVserological diagnostic reagent in next work.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第2期60-65,共6页
China Biotechnology
基金
"十一五"国家科技支撑计划(2006BAD06A14)资助项目
