犬瘟热核衣壳蛋白原核表达及蛋白纯化

Prokaryotic Expression and Protein Purification of Canine Distemper Nucleocapsid Protein

犬瘟热病毒(CDV)是一种单链RNA病毒,作为转录因子合成蛋白,共编码六个结构蛋白,其中核衣壳蛋白N(CDV-N)是保守性较强的免疫原性蛋白,在病毒感染时能引起强烈的抗体反应,对于CDV的诊断具有非常重要的价值。本研究的目的是构建CDV-N蛋白原核表达载体,利用原核表达技术纯化CDV-N蛋白。利用DNAStar软件预测CDV-N蛋白抗原表位,选择588bp截短体用于原核表达。设计引物,以本实验室保存的pMD18-T-CDV质粒为模板,PCR扩增CDV-N588片段,并将其TA克隆至pMD18-T载体。通过酶切鉴定和DNA测序,从pMD18-T-CDV-N588质粒上切取CDV-N588片段,再将其亚克隆至pET30a原核表达载体,构建重组质粒pET30a-CDV-N588。该重组质粒转化大肠杆菌BL21,ITPG在37℃诱导表达His-CDV-N588融合蛋白,并用SDS-PAGE和Western Blotting加以验证。相同条件下大量增菌诱导,Ni-NTA琼脂糖珠在变性条件下分离纯化His-CDV-N588融合蛋白,再次用SDS-PAGE和Western Blotting进行验证。结果表明:pET30a-CDV-N在大肠杆菌BL21中得到高效表达;SDS-PAGE和Western Blotting检测证实,我们成功纯化了His-CDV-N588融合蛋白。本研究为制备CDV-N588蛋白单克隆抗体和研制胶体金诊断试纸条奠定了基础。

As a transcription factor for protein synthesis,Canine distemper virus（CDV）is a highly conserved singlestrand RNA virus.It is composed of six proteins,of which nucleocapsid protein N（CDV-N）is a conservative strong immunogenicity protein,which is very important and valuable for diagnosis of CDV.When virus infection happens to canine,it causes strong antigen-antibody response.This paper was to construct a prokaryotic expression vector for CDV-N,and to purify CDV-N protein using prokaryotic expression technique.By means of DNAStar,the epitope of CDV-N was predicted,and a truncated CDV-N（588 bp）was selected for prokaryotic expression.A pair of primers were designed,and the truncated CDV-N588 was amplified by PCR form the plasmid pMD18-T-CDV,which was stored in our laboratory,and subsequently cloned to a pMD18-T vector.Confirmed by endonuclease digestion and DNA sequencing,the CDV-N588 fragment was removed from the recombinant plasmid pMD18-T-CDV-N588,and then subcloned to a vector pET30 ato construct a recombinant plasmid pET30 a-CDV-N588.The prokaryotic expression plasmid was transformed to E.Coli BL21,His-CDV-N588 fusion protein was induced to express by isopropyl-β-d-thiogalactoside（IPTG）at 37℃,and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）and Western Blotting assay.In the same way,large volume of expressing culture was prepared,and His-CDV-N588 protein was purified using NI-NTA argrose under denaturing condition.Finally,the usion protein was identified by SDS-PAGE and Western Blotting assay.The results showed that the prokaryotic expression plasmid pET30 a-CDV-N588 was efficiently expressed in E.Coli BL21,and His-CDV-N588 fusion protein was successfully purified,which was identified by SDS-PAGE and Western Blotting assay.This paper will lead to preparation of CDV-N monoclonal antibody and its colloidal gold diagnostic test strips.