Rab39A多克隆抗体的制备与鉴定

Preparation and Identification of Rab39A Polyclonal Antibody

制备抗人Rab39A多克隆抗体并进行纯化检测,以用于Rab39A功能的研究.选取免疫原性强且特异性高的羧基端42个氨基酸(176～217aa)作为目的片段.PCR法扩增并构建重组表达质粒pGEX-4T-1-Rab39C42.异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并纯化GST-Rab39C42融合蛋白,免疫新西兰兔,制备多克隆抗体.然后以亲和层析法纯化抗体,利用Western blot和免疫荧光检测抗体的特异性.结果表明,构建的原核表达载体经IPTG诱导后高效表达GST-Rab39C42蛋白,纯化后抗体质量浓度为1μg/μL;Western blot实验显示,抗体可以识别外源Rab39A蛋白和多个细胞系的内源Rab39A蛋白;抗体中和实验和RNA干扰实验证明了该抗体具有特异性.本研究成功构建了pGEX-4T-1-Rab39C42原核表达载体,并制备了特异性的抗人Rab39A兔多克隆抗体,为进一步研究Rab39A的功能提供了有力的支持.

In order to study the function of small OTPase homo Rab39A,the specific polyclonal antibody against Rab39A was genera- ted. The fragment of 42 amino acids at the carboxyl terminal（176-217aa）of Rab39A, with potential strong antigenicity, was amplified through PCR and sub-cloned into pGEX 4T-1 vector to generate pGEX-4T 1-Rab39C42 expression construct. The pGEX-4T-1- Rab39C42 expression construct was induced and expressed in Escherichia coli. DH5c~ with IPTG. Then the affinity purified GST- Rab39C42 was used to immunize the New Zealand White Rabbits for generating polyclonal antibody. The antibody was purified through affinity chromatography method, the specificity of the antibody was characterized. The results showed that the recombinant GST-Rab39C42 fusion protein was efficiently expressed, and it could be used as antigen. The concentration of the purified antibody was up to 1 μg/μL. Western-blot experiments demonstrated that the antibody could recognize both the exogenously expressed Rab39A and the endogenous Rab39A in different cell lines. The specificity of the antibody was further proved by neutralization exper- iment and RNAi experiment. Thus the specific rabbit polyclonal antibody against human Rab39A was successfully generated, which providing strong support for further investigation of biological function of Rab39A.