A型塞内卡病毒VP1蛋白的原核表达及纯化  

Prokaryotic Expression and Purification of Seneca Virus A VP1 Protein

作　　者：杜文琪 夏立叶 李桂梅 单虎 DU Wenqi;XIA Liye;LI Guimei;SHAN Hu(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Novel Veterinary Pharmacy Innovation Center of Shandong Province,Qingdao 266109,China;Research Center for Engineering Technology in Veterinary Medicine and Veterinary Diagnostic Reagent of Shandong Province,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,青岛266109 [2]山东省新兽药创制协同创新中心,青岛266109 [3]山东省兽药诊断试剂工程技术研究中心,青岛266109

出　　处：《中国畜牧兽医》2022年第7期2708-2715,共8页China Animal Husbandry & Veterinary Medicine

基　　金：山东省生猪产业技术体系(SDAIT-08-07)。

摘　　要：【目的】实现A型塞内卡病毒(Seneca virus A,SVA)VP1蛋白在大肠杆菌中的高效可溶性表达,以建立SVA抗体液相芯片技术检测方法。【方法】根据GenBank收录的SVA VP1基因序列(登录号:KY747514.1),基于大肠杆菌的密码子偏好性优化合成VP1基因,克隆到pCold TF载体,构建重组质粒pCold TF-VP1。将重组质粒pCold TF-VP1转化大肠杆菌BL21(DE3)感受态细胞,进行IPTG诱导表达。通过优化诱导时间及IPTG浓度得出最佳诱导表达条件。采用His标签镍柱纯化VP1重组蛋白,将纯化后的SVA VP1蛋白与荧光微球进行偶联,偶联好的微球与阴阳性血清反应,利用Luminex 200系统上机检测背景及阴阳性血清的中值荧光强度(MFI),从而判断阴阳性,以用于猪血清中SVA抗体的检测。【结果】重组质粒pCold TF-VP1成功转入大肠杆菌BL21感受态细胞,并以可溶性蛋白形式表达。经诱导表达在约82 ku处出现阳性条带。当重组菌株诱导条件为16℃、IPTG终浓度为1.2 mmol/L、诱导时间为3 h时,VP1蛋白表达量最高;经Western blotting分析,可在82 ku处出现明显条带。将VP1蛋白与荧光微球偶联,阴性对照(背景)的平均荧光值为17(<100)。测试孔的平均MFI为2339.5(>2000),证明SVA VP1蛋白与微球偶联成功,偶联成功的荧光微球可用于检测猪血清中SVA抗体的检测。【结论】本研究在大肠杆菌中成功表达并纯化了SVA VP1蛋白,初步建立了SVA抗体液相芯片检测方法,为后续研究奠定了基础。【Objective】This study was aimed to express Seneca virus A(SVA)VP1 protein in Escherichia coli(E.coli),and establish the detection method of SVA antibody by liquid chip technology.【Method】According to the SVA VP1 gene sequence(KY747514.1)from GenBank,the VP1 gene was synthesized based on the codon preference of E.coli,and cloned into pCold TF vector to construct the recombinant plasmid pCold TF-VP1.The pCold TF-VP1 was transformed into E.coli BL21(DE3)competent cells and induced by IPTG.The inducing conditions were studied by optimizing the induction time and IPTG concentration.Recombinant VP1 protein was purified by nickel column.The purified SVA VP1 protein was coupled with fluorescent microspheres,and the microspheres were reacted with SVA negative and positive serum.Luminex 200 system was used to detect the background and the median fluorescence intensity(MFI)of the negative and positive serum.【Result】The recombinant plasmid pCold TF-VP1 was successfully transformed into E.coli BL21 competent cells and expressed as soluble protein with the molecular weight of 82 ku.The VP1 protein was expressed at the highest yield when inducing at 16℃for 3 h and the final concentration of IPTG was 1.2 mmol/L.The average MFI of negative control was 17(<100).The average MFI of the test microspheres was 2339.5(>2000),indicating that SVA VP1 protein was successfully coupled with the microspheres.The coupled fluorescent microspheres could be used to detect SVA antibody in porcine serum.【Conclusion】VP1 protein of SVA was successfully expressed in E.coli.The expression conditions were optimized,the target protein was purified and could be used to develop a liquid chip analysis for detection of SVA antibody.

关 键 词：A型塞内卡病毒(SVA) VP1蛋白 大肠杆菌 蛋白纯化 可溶性表达 液相芯片 

分 类 号：S852.651[农业科学—基础兽医学]