PEDV结构蛋白S和M在昆虫细胞中的表达及病毒样颗粒鉴定  

Expression and identification of virus-like particles of S and M proteins of porcine epidemic diarrhea virus in insect cells

作　　者：秦艺文 雷昕诺 王东亮 杨文兵 雷波 余婉婷 王乃东 QIN Yi-wen;LEI Xin-nuo;WANG Dong-liang;YANG Wen-bing;LEI Bo;YU Wan-ting;WANG Nai-dong(College of Veterinary Medicine,Hunan Agricultural University,Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines,Research&Development Center for Animal Reverse Vaccinology of Hunan Province,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院兽用蛋白质工程疫苗湖南省重点实验室兽用疫苗逆向创制湖南省工程研究中心,湖南长沙410128

出　　处：《中国兽医科学》2022年第2期205-213,共9页Chinese Veterinary Science

基　　金：湖南省自然科学基金面上项目(2018JJ2177);湖南农业大学“双一流”建设项目(SYL2019048)。

摘　　要：为在昆虫细胞中表达猪流行性腹泻病毒(PEDV)的S和M蛋白,并探讨S和M蛋白能否自动组装成病毒样颗粒(VLPs)分泌至培养基中,本研究构建了3种不同的重组质粒(pFastBac-S、pFastBac-M和pFastBac-S-M),转化入DH10Bac感受态细胞,经蓝白斑筛选及PCR鉴定获得重组Bacmid,通过转染昆虫细胞Sf9获得重组杆状病毒(BV-S、BV-M和BV-S-M)。在悬浮培养的昆虫细胞Sf9中进行表达,表达分泌的上清通过蔗糖梯度离心进行浓缩纯化,最终获得分泌型的S-MVLPs。通过Western-blotting分析S和M蛋白的表达和分泌特性,透射电镜观察VLPs形态,间接ELISA检测VLPs与猪PEDV阳性血清的反应性,最后免疫小鼠后检验其免疫原性。结果显示,S和M蛋白均能以单独或共表达的形式(S-M)分泌至培养基中。电镜下可观察到由S-M蛋白形成的直径约为90~190 nm的VLPs。间接ELISA结果显示,S、M和S-M蛋白可与PEDV猪阳性血清发生特异性反应,免疫小鼠3次后可产生高水平特异性Ig G抗体。以上结果表明,在Sf9昆虫细胞中共表达S和M蛋白可以获得分泌型S-MVLPs,并具有良好的免疫原性。In order to express S and M protein of porcine epidemic diarrhea virus in insect cells,and to explore whether the S and M protein could assemble automatically into VLPs and secrete into culture medium.In this work,three recombinant plasmids(p Fast Bac-S,p Fast Bac-M and p Fast Bac-S-M)were constructed,then the plasmids were transformed into competent cells DH10 Bac,and positive Bacmid DNA was screened through the blue-white screening and verified by PCR.The recombinant baculovirus(BV-S,BV-M and BV-S-M)were obtained by transfected insect cells Sf9 with recombinant bacmids.Recombinant proteins were expressed in Sf9 suspension culture,and the culture medium were purified by sucrose gradient centrifugation,in order to obtain secretory S-M VLPs.The expression and secretion properties of S and M protein were analyzed by Western-blotting,and the purified product was observed under TEM,the antigenicity and immunogenicity were tested by ELISA.The Western-blotting results showed that S and M protein secreted from Sf9 into culture medium through expression alone or co-expression(S-M).S-M VLPs with diameters in 90—190 nm were observed under TEM.ELISA results indicated that S,M and S-M protein can be detected by PEDV positive pig serum.After three times of vaccination,high level of specific Ig G was boosted in mice.This study showed that S-M VLPs can secreted from Sf9 into culture medium and demonstrated strong immunogenicity.

关 键 词：猪流行性腹泻病毒(PEDV) 结构蛋白 重组杆状病毒 SF9细胞 病毒样颗粒 

分 类 号：S852.659.6[农业科学—基础兽医学]