摘要
将PCR扩增的鸡CD4基因片段克隆到pET-32a原核表达载体,构建获得原核表达重组载体pET-32a-CD4。重组载体导入大肠杆菌BL21(DE3),经过IPTG诱导表达,Ni-NTA亲和树脂纯化获得融合蛋白His-CD4。以此融合蛋白为抗原免疫BALB/c小鼠,用杂交瘤细胞技术制备了1株能够稳定传代并分泌抗CD4多肽单抗的杂交瘤细胞株,命名为3E12。经检测该抗体亚类为IgG2b,单抗腹水的间接ELISA效价为1:105,Westernblot结果显示单抗与CD4多肽能特异性地结合。
The gene fragment of chicken CD4 was obtained by PCR and was cloned into pET-32a to construct recombinant prokaryotic expression vector pET-32a-CD4.And then the recombinant vector was transformed into Escherichia coli BL21（DE3）.Fusion protein His-CD4 was induced by IPTG and purified with affinity Ni-charged resin.A hybridoma cell line secreting monoclonal antibody（McAb） against polypeptide CD4 was produced by fusing mouse myeloma cells（SP2/0） with spleen cells from BALB/c mice immunized with the recombinant protein,which was named as 3E12.The McAb was belonged to IgG2b subtype.The titer of ascitic fluids of the McAb was 1：105 in indirect ELISA,and showed specific reaction to polypeptide CD4 in Western blot.
出处
《中国家禽》
北大核心
2011年第8期22-24,共3页
China Poultry
基金
国家自然基金项目(30671537)
关键词
鸡
CD4
原核表达
单抗
chicken
CD4
prokaryotic expression
monoclonal antibody
