猪A群轮状病毒VP6蛋白单克隆抗体的制备及抗原表位鉴定
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摘要
为制备猪A群轮状病毒(RV)VP6蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究以原核表达、纯化的重组VP6蛋白免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0细胞进行融合,r VP6-GST为包被抗原,通过间接ELISA筛选出一株稳定分泌抗VP6蛋白的MAb杂交瘤细胞株(1F4)。MAb鉴定结果显示其抗体亚类为Ig G1型,轻链为κ链;细胞上清液及腹水效价分别为1∶12 800和1∶106。Western blot和间接免疫荧光结果显示该MAb能够与天然的VP6蛋白反应。应用肽扫描技术鉴定显示该MAb对应抗原表位核心序列为134DYIENWNLQNR144。该MAb的制备为进一步研究VP6蛋白功能和建立RV检测方法奠定基础。
To prepare the monoclonal antibody(MAb) against VP6 protein of group A porcine rotavirus(RV) and identify the antigenic epitope, a hybridoma stable secreting MAb against VP6 protein was prepared by fusing SP2/0 cells with spleen cells from BALB/c mice immunized with recombinant VP6 protein. Identification of the MAb indicated that it belonged to Ig G1 subtype withκ chain. The titers in cell culture medium of the hybridoma and ascites were 1 ∶12 800 and 1 ∶106, respectively. In addition, the MAb was positively reacted with authentic VP6 protein of porcine RV detected by western blot and indirect immunofluoresence assay. Moreover, the antigenic epitope sequence of134DYIENWNLQNR144 was identified by the pepscan technique. Those results provided a basis for the further study on the function of VP6 and development of diagnostic method.
To prepare the monoclonal antibody(MAb) against VP6 protein of group A porcine rotavirus(RV) and identify the antigenic epitope, a hybridoma stable secreting MAb against VP6 protein was prepared by fusing SP2/0 cells with spleen cells from BALB/c mice immunized with recombinant VP6 protein. Identification of the MAb indicated that it belonged to Ig G1 subtype withκ chain. The titers in cell culture medium of the hybridoma and ascites were 1 ∶12 800 and 1 ∶106, respectively. In addition, the MAb was positively reacted with authentic VP6 protein of porcine RV detected by western blot and indirect immunofluoresence assay. Moreover, the antigenic epitope sequence of134DYIENWNLQNR144 was identified by the pepscan technique. Those results provided a basis for the further study on the function of VP6 and development of diagnostic method.
引文
[1]时洪艳,陈建飞,王承宝,等.猪轮状病毒Rotavirus A pig/China/NMTL/2009/G9P[23]株的分离与鉴定[J].中国预防兽医学报,2011,33(9):681-684.
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[7]张志榜,陈建飞,时洪艳,等.猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报,2011,33(7):568-570.
[8]Jones S L,Cox J C,Pearson J E.Increased monoclonal antibody ascites production in mice primed with Freund's incomplete adjuvant[J].J Immunol Methods,1990,129(2):227-231.
[9]Garaicoechea L,Olichon A,Marcoppido G,et al.Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice[J].J Virol,2008,82(29):9753-9764.
[10]Kohli E,Muurice L,Bourgeois C,et al.Epitopemapping of the major inner capsid protein of group A rotavirus using peptide synthesis[J].Virology,1993,194(1):110-116.
[2]徐璐,黄小波,文心田,等.A组轮状病毒蛋白研究进展[J].中国人兽共患病学报,2007,23(7):729-731.
[3]Parashar U D,Gibson C J,Bresse J S,et al.Rotavirus and severe childhood diarrhea[J].Emerg Infect Dis,2006,12:304-306.
[4]时洪艳,冯力,陈建飞,等.猪轮状病毒OSU强毒株vp7基因的克隆及其抗原表位原核表达和免疫学活性分析[J].中国预防兽医学报,2008,30(2):136-140.
[5]高慎阳,查恩辉,王坤.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
[6]迟延彬,陈建飞,时洪艳,等.A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定[J].中国畜牧兽医,2014,41(2):70-74.
[7]张志榜,陈建飞,时洪艳,等.猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报,2011,33(7):568-570.
[8]Jones S L,Cox J C,Pearson J E.Increased monoclonal antibody ascites production in mice primed with Freund's incomplete adjuvant[J].J Immunol Methods,1990,129(2):227-231.
[9]Garaicoechea L,Olichon A,Marcoppido G,et al.Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice[J].J Virol,2008,82(29):9753-9764.
[10]Kohli E,Muurice L,Bourgeois C,et al.Epitopemapping of the major inner capsid protein of group A rotavirus using peptide synthesis[J].Virology,1993,194(1):110-116.