摘要
为获得牛病毒性腹泻病毒(BVDV)E2蛋白的多克隆抗体,本研究截短了BVDV E2氨基端前600个碱基,并构建原核表达载体pET28a-E2,优化E2序列并将重组载体转化到BL21感受态细胞中。37℃、1 mmol/L IPTG条件下诱导4 h,SDS-PAGE凝胶电泳观察在15 kDa处表达目的蛋白。Ni-NTA亲和层析纯化E2蛋白并免疫小鼠制备鼠抗血清。以获得的血清为一抗,Western blot检测pET28a-E2在BL21中表达的总蛋白,证明免疫性良好。利用BVDV感染MDBK细胞,Western blot成功检测到MDBK细胞表达的E2蛋白,证明E2抗体具有良好的特异性。ELISA检测E2多克隆抗体的效价达1∶128 000。本研究成功制备出BVDV E2多克隆抗体,为进一步研究该蛋白的功能及BVDV活载体疫苗的鉴定提供了技术和材料支持。
In order to obtain the polyclonal antibody against bovine viral diarrhea virus E2 protein,in this study,the first 600 bp of the amino terminal of BVDV E2 was truncated and the prokaryotic expression vector pET28a-E2 was constructed,the E2 sequence was optimized,and the recombinant vector was transformed into BL21 capable cells.The target protein was induced at 37℃ and 1mmol/L IPTG for 4 h,SDS-PAGE gel electrophoresis was used to observe the expression of target protein at 15 kDa.Ni-NTA affinity chromatography was used to purify protein and immunize mice to prepare anti-serum.The prepared serum was used as the primary antibody,Western blot to detect the total protein expressed by pET28a-E2 in BL21,which proved that the immunity was good.Using BVDV to infect MDBK cells,Western blot successfully detected the E2 protein expressed by MDBK cells,which proved that the prepared had good specificity.The titer of polyclonal antibody against E2 was 1:128 000 by ELISA.This study successfully prepared the corresponding E2 polyclonal antibody,which provided technical and material support for further study of the function of the protein and the identification of BVDV live vector vaccine.
作者
李胜文
王炜
赵文栋
丁乃峥
王洪梅
何洪彬
何成强
LI Shengwen;WANG Wei;ZHAO Wendong;DING Naizheng;WANG Hongmei;HE Hongbin;HE Chengqiang(Shandong Key Laboratory of Animal Resistance,College of Life Sciences,Shandong Normal University,Jinan 250014,China)
出处
《中国动物传染病学报》
CAS
北大核心
2023年第4期189-194,共6页
Chinese Journal of Animal Infectious Diseases
基金
山东省重点研发项目(2017GNC10125,2019GSF107020)。
关键词
牛病毒性腹泻病毒
E2基因
原核表达
多克隆抗体
Bovine viral diarrhea virus
E2gene
prokaryotic expression
polyclonal antibodies
