PNAS-2蛋白单克隆抗体的制备和初步鉴定

Preparation and Identification of Monoclonal Antibody against PNAS-2 Protein

本研究制备凋亡相关蛋白PNAS-2的单克隆抗体,并对其进行初步鉴定,为PNAS-2蛋白功能研究提供必需工具。以PNAS-2的合成肽为免疫原,通过动物免疫、细胞融合、克隆化制备抗PNAS-2蛋白的单克隆抗体,并用Western blot、免疫荧光、ELISA实验对单克隆抗体的特异性、效价、亚型进行了检测。结果表明:获得了稳定的分泌型杂交瘤细胞株S-31-7,属IgG1λ亚型,腹水效价为1:8000;Western blot测得分子量为28kD的单条特异性条带;免疫荧光实验显示胞浆中有阳性反应。结论:制备的单克隆抗体特异性好、效价高,能够作为深入研究PNAS-2蛋白功能的有利工具。

This study was purposed to prepare and primarily identify the specific monoclonal antibodies （McAbs） against the apoptosis related protein PNAS-2 so as to provide the essential tool for study of PNAS-2 function. The McAbs against PNAS-2 were prepared via the immunization of mice, cell fusion and cloning using synthetic peptide of PNAS-2 as immunogen ; the specificity, titer and subtype of McAb were detected by Western blot, ELISA and immunofluorescence. The results showed that the stable hybridoma cell line S-31-7 producing McAbs against PNAS-2 protein was successfully obtained. The immunoglobulin of the McAb was identified to be IGgl h. The titer of ascetic fluid fled McAb were 1 ： 8 000. A single specific band with 28 kD was shown in Western blot test, and the antigen recognized was present in cell cytoplasm by immunofluorescence. In conclusion, the obtained McAb against PNAS-2 displays strong specificity and high titer, which may be applied to the advanced research on PNAS-2 protein.