鸭MAPK1蛋白的原核表达和单克隆抗体的制备

Prokaryotic expression and monoclonal antibody preparation of duck MAPK1

为研制鸭丝裂原活化蛋白激酶1(MAPK1)单克隆抗体,从樱桃谷鸭脾样品扩增鸭MAPK1基因,将其克隆至原核表达载体pET-28a(+)中,构建鸭MAPK1基因原核表达质粒,诱导表达重组蛋白,经纯化后作为免疫原免疫BALB/c小鼠。经过3次免疫后,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞进行融合,用间接ELISA筛选分泌抗鸭MAPK1的单克隆杂交瘤细胞株,进行3次亚克隆纯化后,制备小鼠腹水。采用间接ELISA测定腹水效价,用Western-blot检测其特异性,并进行单克隆抗体的亚类鉴定。测序结果表明,鸭MAPK1基因编码区全长1 107bp,编码369个氨基酸。SDS-PAGE检测结果表明,本研究获得了可溶性的42ku的鸭MAPK1重组蛋白。用表达的重组蛋白免疫BALB/c小鼠后,得到2株抗鸭MAPK1的阳性杂交瘤细胞株,腹水效价均达到1∶12 800以上,均为IgG1亚类。Western-blot分析结果表明,制备的单克隆抗体具有良好的免疫反应特异性。以上结果表明,本研究成功制备了抗鸭MAPK1的单克隆抗体,为进一步研究其在先天免疫反应中的作用奠定了基础。

The objective of this study is preparation of monoclonal antibodies(McAbs)against duck MAPK1.Duck MAPK1 gene was amplified from the spleen of Cherry Valley Duck,and cloned into plasmid pET-28a(+)to construct pET-MAPK1.The recombinant protein was expressed,purified and used as an antigen for immunization.BALB/c mice were immunized for 3times and the hybridoma technique was performed for the McAb development.Hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay and positive clones were subjected to subclone for three times.The subtypes of the McAbs were analyzed.Sequence analysis indicated that duck MAPK1 gene was 1 107 bp in length and encoded 369 amino acids.SDS-PAGE analysis showed a fusion protein band of 42 ku.The established two hybridoma cell clones(4A2and 28D7)produced stably anti-MAPK1 McAb.The isotype of both McAbs was identified as IgG1.Western-blot analysis showed that both McAbs reacted specifically with duck MAPK1.The two high titers of McAbs were prepared successfully,which could be used for further study of MAPK1 in duck innate immunity.