glipr1基因的原核表达及多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of glipr1 gene

目的克隆表达胶质瘤致病相关蛋白1基因(glipr1)并制备多克隆抗体,运用该抗体检测各肿瘤细胞系中GLIPR1的表达。方法通过PCR技术以含glipr1基因的c DNA克隆质粒(p LX304-glipr1)为模板获得glipr1(M)片段,再将此片段克隆至表达载体p ET-15b上,并转入大肠杆菌BL21-Codon Plus(DE3)进行表达;Ni柱纯化后的GLIPR1(M)蛋白作为抗原免疫家兔,获得多克隆抗体,Western blot法检测其特异性,并检测GLIPR1在A549、PC14、LNCa P、PC3及U87细胞中的表达。结果成功构建了表达载体p ET-15bglipr1(M),并获得了纯化的GLIPR1(M)蛋白,成功制备了兔抗GLIPR1多克隆抗体,特异性高,可用于检测各细胞系中GLIPR1的表达,GLIPR1在U87中的表达最高。结论成功表达了GLIPR1(M)蛋白,并获得了多克隆抗体,为进一步研究GLIPR1的功能和作用机制奠定了基础。

Objective To clone and express glioma pathogenesis-related protein 1 gene, and prepare the anti- GLIPR1 polyclonal antibody. Use anti-GLIPR1 to check the expression of GLIPR1 in some cancer cell lines. Methods cDNA clone plasmids （ pLX304-gliprl ） were used as template to amplify the gliprl （M） fragment by PCR. The fragment was cloned into the pET-15b vector and expressed in E. coli BI21-CodonPlus（DE3）. The GLIPR1 （M） protein was purified by Ni affinity chromatography and was used as antigen to prepare poXyclonal antibody. Western blot analysis was used to check the specificity of the antibody and detect the expression of GLIPR1 in A549, PC14, LNCaP, PC3 and U87 ceils. Results Expression vector pET-15b-gliprl （M） was successfully constructed, and the purified GLIPR1 （M） protein was obtained, the polyclonal anti-GLIPR1 antibody was successfully prepared and could be used to check the expression of GLIPR1 in cancer cell lines. GLIPR1 was highly expressed in U87 cells. Conclusion The GLIPR1 （M） protein and the polyclonal anti-GLIPR1 antibody are successfully prepared, which lays the foundation for the research of GLIPR1 function mechanism.