VMP1基因的克隆及其抗体的制备与鉴定

为了得到效价高、特异性强、能用于检测内源性VMP1蛋白的抗体，采用RT-PCR法，从L929细胞株中克隆鼠VMP1基因，重组入pCMVS载体，用PCR扩增与其氨基酸132-239区域对应的DNA片断，将该片段连接到原核表达载体pGST parallel中，在大肠杆菌BL21菌株中大量表达，经谷胱甘肽-琼脂糖树脂纯化后，得到高纯度的VMP1抗原．免疫新西兰兔，获得抗VMP1蛋白的兔抗血清，将血清纯化后即得到抗VMP1的多克隆抗体．用Western blot分析手段检测了该抗体的特异性及效价．在用该抗体检测外源性和内源性VMP1蛋白的试验中，证实该抗体效价高，特异性强，效果很理想．此方法可用于获得大量廉价的抗VMP1蛋白的高滴度和高特异性的抗体，为进一步研究VMP1在细胞内，尤其是在信号转导通路中的功能和作用奠定了基础．

Preparation and Identification of the Rabbit Polyclonal Antibodies from VMP1

To get high concentrative antibody, specifically for Western blot of VMP1, VMP1 gene was cloned from the L929 cell line by RT-PCR and recombined into the vector of pCMV5-myc. PCR was used to amplify the DNA fragment corresponding to the amino acid 132-239 sequence of VMP1 from pCMV5-myc-VMP1. Then the fragment was ligased to pGST-parallel vector, overexpressed in The E coli. strain BL21. The protein was purified by GST beads, then immuned rabbits. 9 weeks later, the serum of rabbits was harvested and anti-VMP1 polyclonal antibodies was purified. VMP1 was successfully detected by western blot with anti-VMP1 antibody in 293T cells transfected with pCMV5-myc-VMP1 and endogenously in mouse L929 cell line. The results show that the antibody is high concentrative with good immunological characteristics. Antibody with high concentration and specificity can be well obtained by this method, which most labs can afford. Furthermore, the antibody lays a good foundation for further research on the function of VMP1 in cell, especially on its role in signal transduction pathway.