鸡宿主限制因子SAMHD1单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody of Chicken Host Restriction Factor SAMHD1

为探讨鸡源宿主限制因子SAMHD1(chSAMHD1)的功能及作用机制,研究将前期制备并纯化的chSAMHD1原核表达蛋白免疫8周龄BALB/c小鼠,应用细胞融合技术筛选单克隆抗体杂交瘤细胞株,制备抗chSAMHD1单克隆抗体。结果显示:经间接ELISA筛选和2次亚克隆最终获得了5株能稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为1A8-2-1、1A12-1-8、2B9-7-6、1F5-1-4、1F7-7-7,小鼠腹水效价较高的1A8-2-1和2B9-7-6抗体亚型均为IgG1;间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)鉴定结果均表明,单克隆抗体1A8-2-1可特异性识别293T细胞和DF-1细胞过表达的chSAMHD1蛋白。该研究成功获得了特异性抗chSAMHD1单克隆抗体,可用于ELISA、IFA和Western blot检测,为鸡源SAMHD1蛋白生物学功能的研究奠定基础。

In order to study the function and mechanism of chicken host restriction factor SAMHD1 protein and prepare chicken source SAMHD1(chSAMHD1) monoclonal antibody, in this study, 8-week-age BALB/c mice were immunized with prokaryotic expression protein chSAMHD1 prepared in the previous stage. Monoclonal antibody hybridoma cell lines were screened by cell fusion technology to prepare anti-chSAMHD1 monoclonal antibody. The results showed that five hybridoma cell lines with stable monoclonal antibody secretion were finally obtained by indirect ELISA and2 subclones, named 1 A8-2-1, 1 A12-1-8, 2 B9-7-6, 1 F5-1-4, and 1 F7-7-7, respectively. Ascitic fluid was prepared by 1 A8-2-1 and 2 B9-7-6 monoclonal antibodies with high titer, and both monoclonal antibody subtypes were identified as IgG1.And the results of indirect immunofluorescence(IFA)and Western blot showed that monoclonal antibody 1 A8-2-1 could specifically recognize chSAMHD1 protein over-expressed in 293 T cells and DF-1 cells. The results showed that the specific monoclonal antibody against chSAMHD1 was successfully obtained, which could be used for ELISA, IFA and Western blot analysis, laying a foundation for further study on the biological function of chicken SAMHD1 protein.