鼠抗人ANKRD13C单克隆抗体的制备与鉴定

Preparation and characterization of mouse monoclonal antibody against human ANKRD13C

为制备针对锚蛋白重复结构域蛋白13C(ankyrin repeat domain-containing protein 13C,ANKRD13C)的单克隆抗体,利用PCR扩增人源ANKRD13C基因,并与原核表达载体pET-32a(+)和pGEX-6P-1连接构建重组质粒,通过诱导表达纯化ANKRD13C重组蛋白、免疫小鼠、PEG法融合细胞和单克隆化,获得9株分泌特异性单克隆抗体的杂交瘤细胞株,包括7株IgG1类抗体和2株IgM类抗体。经免疫印迹法鉴定,其中1株IgG1类抗体8E3能特异性识别细胞内源性表达的ANKRD13C,将该杂交瘤细胞注射入小鼠腹腔内生产腹水,纯化后经ELISA检测其效价为1:2.51×10^(5),亲和常数达(9.78±0.55)×10^(9) L·mol^(-1)。这一研究表明,该抗体可应用于ELISA、Western blot和IFA等试验,为深入研究ANKRD13C生物学功能奠定基础。

In order to prepare a monoclonal antibodies against ankyrin repeat domain-containing protein 13C(ANKRD13C)in this study,the ANKRD13C gene was cloned by PCR and cloned into the prokaryotic expression vectors pET-32a(+)and pGEX-6P-1 to construct the recombinant plasmids.Through induced expression and purification of ANKRD13C recombinant proteins,immunizing mice,PEG-method fusion cells and cloning of hybrid cells,nine hybridoma cell lines secreting specific monoclonal antibodies were obtained,including seven IgG1 class antibodies and two IgM class antibodies.The results showed that 8E3,one of IgG1 antibodies against ANKRD13C,specifically recognized endogenous ANKRD13C by Western blot,and the hybridoma cells were injected into the abdominal cavity of mice to produce ascites.After purification,the titer measured by ELISA was 1:2.51×10^(5),the affinity constant reached(9.78±0.55)×10^(9) L·mol^(-1).The research showed that 8E3 can be applied in ELISA,WB and IFA experiments,and lays the foundation for further research on the biological roles of ANKRD13C.