口蹄疫病毒3C蛋白多克隆抗体的制备与鉴定
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摘要
扩增口蹄疫病毒(FMDV) 3C蛋白编码基因,将其克隆到原核表达载体,并转化至大肠杆菌BL21中进行诱导表达; 3C重组蛋白纯化后免疫新西兰大白兔,制备多克隆抗体,通过ELISA和Western blot检测多克隆抗体的效价和特异性,并将该抗体通过间接免疫荧光(IFA)和Western blot应用于3C的检测。ELISA结果显示,制备的3C蛋白多克隆抗体效价达1∶256 000; IFA结果显示,该抗体能够检测到3C蛋白在细胞中的定位; Western blot结果显示,该多克隆抗体能够检测到在细胞中过表达的3C蛋白。本研究制备的FMDV 3C蛋白多克隆抗体为FMDV及其3C蛋白的相关研究提供了重要的物质基础。
In this study,the FMDV 3C gene was amplified and cloned into a p ET-28 a vector to construct the prokaryotic expression plasmid p ET-28a-3C,which was further transformed into the Escherichia coli BL21 to induce expression. We used Ni-NTA agarose affinity chromatography and Millipore ultrafiltration tube to purify the 3C fusion protein. Then,New Zealand white rabbits were immunized with the purified recombinant protein to produce polyclonal antibodies. ELISA was used to detect the titer and Western blotting was used to analyze the specificity of the polyclonal antibody. The polyclonal antibody was further applied to detect the eukaryotic expression of the 3C protein by IFA and Western blotting. The results of ELISA showed that the titer of the 3C protein polyclonal antibody was as high as 1: 256 000. The polyclonal antibody detected the localization of the 3C protein in cells by IFA. The over-expressed 3C protein in cells could also be detected by Western blotting using the polyclonal antibody. The successful preparation of FMDV 3C polyclonal antibody here provided an important test tool for relevant study of FMDV and its 3C protein.
In this study,the FMDV 3C gene was amplified and cloned into a p ET-28 a vector to construct the prokaryotic expression plasmid p ET-28a-3C,which was further transformed into the Escherichia coli BL21 to induce expression. We used Ni-NTA agarose affinity chromatography and Millipore ultrafiltration tube to purify the 3C fusion protein. Then,New Zealand white rabbits were immunized with the purified recombinant protein to produce polyclonal antibodies. ELISA was used to detect the titer and Western blotting was used to analyze the specificity of the polyclonal antibody. The polyclonal antibody was further applied to detect the eukaryotic expression of the 3C protein by IFA and Western blotting. The results of ELISA showed that the titer of the 3C protein polyclonal antibody was as high as 1: 256 000. The polyclonal antibody detected the localization of the 3C protein in cells by IFA. The over-expressed 3C protein in cells could also be detected by Western blotting using the polyclonal antibody. The successful preparation of FMDV 3C polyclonal antibody here provided an important test tool for relevant study of FMDV and its 3C protein.
引文
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[11]Mayr G A,O’Donnell V,Chinsangaram J,et al.Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus-FMDVconstructs[J].Vaccine,2001,19(15-16):2152-2162.
[12]Ivana S,Valeria Q,Cecilia L,et al.Noveldendrimeric peptide:Immune response and protection against foot-and-mouth disease virus (FMDV) induced by synthetic peptides in sattle[J].Front Immunol,2015,05.00237.
[13]Mc Coy A J,Grosse-Kunstleve R W,Storoni L C.Likelihood-enhanced fast translation functions[J].Acta Crystallogr D Biol Crystallogr,2005,61:458-464.
[14]姚怀兵,赵毅,刘梦丽,等.口蹄疫病毒3C蛋白酶结构与功能及应用研究进展[J].动物医学进展,2017,38(3):102-106.
[15]张启燕,张文会,肖军海,等.3C和3CL蛋白酶及广谱抑制剂的研究进展[J].国际药学研究杂志,2016,43(3):425-430.
[2]南京农业大学.家畜传染病学[M].2版.北京:中国农业出版社,1986:149-159.
[3]王定高.猪口蹄疫的流行特点及防治措施[J].中国畜牧兽医文摘,2015,31(10):135.
[4]吴会杰,汤全发.口蹄疫特点及防治措施探讨[J].中国畜禽种业,2012,8(1):106.
[5]杨彬,柳纪省.口蹄疫病毒3C蛋白酶的结构及功能研究进展[J].动物医学进展,2007,28(9):87-90.
[6]Malirat V,Bergmann I E,de Mendonca Campos R,et al.Molecular epidemiology of foot-and-mouth disease virus type A in South America[J].Vet Microbiol,2012,158:82-94.
[7]周万木.猪口蹄疫的特点及防治措施[J].中国畜禽种业,2015,11(4):48.
[8]Ivana S,Valeria Q,Cecilia L,et al.Novel dendrimeric peptide:Immune response and protection against foot-and-mouth disease virus (FMDV) induce by synthetic peptides in cattle[J].Front Immunol,2015,6.doi:10.3389/conf.fimmu.2015.05.00237.
[9]卢曾军,曹轶梅,刘在新,等.口蹄疫病毒多基因腺病毒穿梭质粒的构建[J].中国兽医科学,2004,34(2):21-24.
[10]田纯见,毕英佐,曹永长,等.口蹄疫病毒3C蛋白酶的T4噬菌体表面展示[J].中国兽医科学,2006,36(7):519-522.
[11]Mayr G A,O’Donnell V,Chinsangaram J,et al.Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus-FMDVconstructs[J].Vaccine,2001,19(15-16):2152-2162.
[12]Ivana S,Valeria Q,Cecilia L,et al.Noveldendrimeric peptide:Immune response and protection against foot-and-mouth disease virus (FMDV) induced by synthetic peptides in sattle[J].Front Immunol,2015,05.00237.
[13]Mc Coy A J,Grosse-Kunstleve R W,Storoni L C.Likelihood-enhanced fast translation functions[J].Acta Crystallogr D Biol Crystallogr,2005,61:458-464.
[14]姚怀兵,赵毅,刘梦丽,等.口蹄疫病毒3C蛋白酶结构与功能及应用研究进展[J].动物医学进展,2017,38(3):102-106.
[15]张启燕,张文会,肖军海,等.3C和3CL蛋白酶及广谱抑制剂的研究进展[J].国际药学研究杂志,2016,43(3):425-430.