中国畜牧兽医2019，Vol.46Issue(1)：256-263,8.DOI:10.16431/j.cnki.1671-7236.2019.01.030
A型塞内卡病毒VP2蛋白原核表达及其多克隆抗体的制备
Prokaryotic Expression of the VP2 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies
摘要
Abstract
The present study was aimed to express the VP2 protein of Senecavirus A (SVA) and prepare its polyclonal antibodies.The full-length coding sequence of the SVAVP2 gene was amplified by RT-PCR using the SVA RNA of GD01/2017 strain as the template.The amplicon was then cloned into the prokaryotic expression vector pET-30a (+) to construct a recombinant plasmid pET-30a-SVA-VP2.After verification by DNA sequencing, the plasmid was transformed into E.coli Rosetta (DE3) competent cells which were then induced by IPTG.The recombinant SVA VP2 protein was purified from the supernatant of the lysate of pET-30a-SVA-VP2plasmid-transformed E.coli Rosetta (DE3) cells using the Ni-NTA agarose under native conditions.The purified VP2 protein was used to immunize the New Zealand White rabbits to prepare its polyclonal antibodies, which were then purified from the rabbit sera by affinity chromatography using theProtein A Sepharose CL-4B.The reactivity of the polyclonal antibodies with SVA was analyzed by an indirect immunofluorescence assay.The results showed that the recombinant SVA VP2 protein was expressed in E.coli Rosetta (DE3) cells in the form of both soluble and inclusion bodies with a molecular weight of about 47 ku, and that the titer of the rabbit anti-VP2 polyclonal antibodies was 1∶64 000.The prepared polyclonal antibodies merely reacted with SVA and had no cross-reactivity with other common porcine pathogens, such as porcine reproductive respiratory and syndrome virus, porcine circovirus type 2, encephalomyocarditis virus, classical swine fever virus and pseudorabies virus.It indicated that the prepared polyclonal antibodies against SVA VP2 protein had good specificity.The successful preparations of recombinant SVA VP2 protein and its polyclonal antibodies provided valuable biomaterials for the establishment of serological detection method for SVA.关键词
A型塞内卡病毒/衣壳蛋白/原核表达/多克隆抗体Key words
Senecavirus A (SVA) /capsid protein/prokaryotic expression/polyclonal antibodies分类
农业科技引用本文复制引用
张永宁,张舟,诸明欣,梅琳,王彩霞,吴绍强,林祥梅..A型塞内卡病毒VP2蛋白原核表达及其多克隆抗体的制备[J].中国畜牧兽医,2019,46(1):256-263,8.基金项目
国家重点研发计划(2016YFD0501102) （2016YFD0501102）
