不同原核表达载体对非洲猪瘟病毒CD2v蛋白可溶性表达及免疫反应性比较  

Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors

作　　者：冯梦珂 王星博[1,2] 林璐璐 崔明仙 颜焰 周继勇[1,2] FENG Mengke;WANG Xingbo;LIN Lulu;CUI Mingxian;YAN Yan;ZHOU Jiyong(Center for Veterinary Sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China;Key Laboratory of Animal Virology of Ministry of Agriculture and Rural Affairs,Hangzhou 310058,Zhejiang,China)

机构地区：[1]浙江大学动物医学中心,浙江杭州310058 [2]农业农村部动物病毒学重点实验室,浙江杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2023年第6期873-880,共8页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金区域创新发展联合基金项目(U21A20256);浙江省重点研发计划项目(2020C02013)。

摘　　要：本研究旨在系统性地探究不同原核表达载体对非洲猪瘟病毒CD2v蛋白的可溶性表达差别,并利用临床非洲猪瘟病毒抗体阳性血清比较包涵体和可溶性CD2v蛋白的免疫反应性。利用5种原核表达载体pCold-TF、pET28a、pMAL-C6T、pGEX-4T-1、pET32a分别表达去除信号肽和跨膜区的非洲猪瘟病毒CD2v蛋白,利用镍-氨三乙酸(nickel-nitrilotriacetic acid,Ni-NTA)亲和层析法分别纯化源自pET28a和pCold-TF表达载体的包涵体和可溶性CD2v蛋白,并用间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)方法比较纯化蛋白的免疫反应性。结果显示,pCold-TF载体以表达可溶性蛋白为主,pMAL-C6T载体同时表达包涵体和可溶性蛋白,而其他载体主要表达包涵体蛋白。临床非洲猪瘟病毒抗体阳性血清检测数据显示,可溶性蛋白的免疫反应性显著优于包涵体蛋白(P<0.05)。pCold-TF载体的触发因子(trigger factor,TF)标签可促进CD2v蛋白的可溶性表达,其表达蛋白的免疫反应性优于包涵体蛋白。本研究结果为CD2v蛋白的免疫原性研究奠定了基础,亦为其他重要抗原的可溶性表达提供了思路。The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus(ASFV)CD2v protein by different prokaryotic expression vectors,and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera.Five prokaryotic expression vectors,namely,pCold-TF,pET28a,pMAL-C6T,pGEX-4T-1 and pET32a,were utilized to express the CD2v protein without the signal peptide and transmembrane region,respectively.The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid(Ni-NTA)affinity chromatography,and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay(ELISA).The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly,while the CD2v protein expressed by the pMAL-C6T vector was insoluble(inclusion body)and soluble,and the CD2v protein expressed by the other vectors was mainly as inclusion body.The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein(P<0.05).The trigger factor(TF)tag of pCold-TF promoted the soluble expression of the CD2v protein,and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein.This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.

关 键 词：非洲猪瘟病毒 CD2v蛋白 原核表达 可溶性表达 免疫反应性 

分 类 号：S855.3[农业科学—临床兽医学]