摘要
为制备用于ELISA检测猪瘟病毒的E^rns蛋白,根据昆虫细胞密码子偏好性对E^rns的基因序列进行优化,将优化后的E^rns基因插入带有信号肽的表达载体pFastBacl-gp67中;随后将重组质粒pFastBacl-gp67-E^rns转化至DH10Bac感受态细胞,经蓝白斑筛选,挑选白斑,提取重组黏粒Bacmid-E^rns;将重组黏粒转染sf21细胞得到杆状病毒,经扩增后得到P 3病毒,利用其大量感染细胞,3 d后收取细胞培养上清;通过镍填料纯化得到分泌表达的E^rns蛋白,通过SDS-PAGE及Western blot检测分泌蛋白的表达水平、纯化情况,用PNGase F处理分析其糖基化水平,通过ELISA分析其抗原性。结果显示,成功构建了重组质粒pFastBacl-gp67-E^rns,蓝白斑筛选获得了重组黏粒Bacmid-E^rns,在此基础上获得了重组杆状病毒,利用其感染sf21细胞,成功分泌表达了E^rns蛋白,纯化所得E^rns蛋白纯度较高,具有较高糖基化修饰水平,且纯化所得E^rns蛋白能特异性识别猪瘟阳性血清,其抗原性良好。
In order to obtain the CSFV E^rns protein for the application in ELISA,the gene sequence of the E^rns protein was optimized according to the codon bias of Spodoptera frugiperda cells.Firstly,the gene was inserted into the recombinant vector pFastBacl-gp67,which contains the gp67 signal peptide sequence.Secondly,the resulting plasmid pFastBacl-gp67-E^rns was transformed into the DH10Bac competent cells to get the recombinant bacmid through blue-white screening.Thirdly,the recombinant Bacmid-E^rns was transfected into the sf21 cells to generate the recombinant baculovirus.Finally,the sf21 cells were infected with the recombinant baculovirus.At three days post infection,culture supernatant was collected.The E^rns protein was purified by nickel-affinity chromatography.The expression level and purity of the protein were identified using SDS-PAGE and Western blot analysis.The glycosylation pattern of the purified E^rns protein was analyzed using PNGase F.And the indirect ELISA was carried out to analyze the antigenicity of the protein.Consequently,the recombinant pFastBacl-gp67-E^rns plasmid was generated.And the Bacmid-E^rns was obtained using blue-white screening.The recombinant baculovirus was used for large-scale expression of E^rns.The secretory expression of E^rns was achieved using signal peptide.It was shown that the purified E^rns was of high purity.And the E^rns was glycosylated properly.The purified E^rns protein could be specifically recognized by the CSFV positive serum,indicating that the purified protein was of high activity.
作者
魏蔷
白怡霖
柴书军
刘运超
宋亚鹏
张改平
WEI Qiang;BAI Yilin;CHAI Shujun;LIU Yunchao;SONG Yapeng;ZHANG Gaiping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences/Henan Key Laboratory of Animal Immunology/Key Laboratory of Animal Immunology,Ministry of Agriculture,Zhengzhou 450002,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)
出处
《河南农业科学》
北大核心
2020年第2期136-141,共6页
Journal of Henan Agricultural Sciences
基金
国家重点研发计划项目(2017YFD0501103)
关键词
猪瘟病毒
E^rns蛋白
分泌表达
糖基化分析
活性
杆状病毒
Classical swine fever virus(CSFV)
E^rns protein
Secretory expression
Glycosylation analysis
Activity
Baculovirus
