小鼠MAdCAM-1分子剪切体的克隆及表达

Cloning and protein expression of mouse MAdCAM-1 spliced variant

目的 获得小鼠粘膜血管定居因子 (MAdCAM 1)分子剪切体cDNA ,并在原核细胞中进行有效表达。方法 从BALB/c小鼠的肠系膜淋巴结中提取总RNA ,利用RT PCR方法获得小鼠MAdCAM 1分子剪切体cDNA ,构建原核表达载体并进行表达 ,纯化融合蛋白制备多抗。结果 核酸序列分析表明获得了正确的小鼠MAdCAM 1分子剪切体cDNA ;小鼠MAdCAM 1剪切体以融合蛋白的形式得到表达 ,主要以包涵体形式存在 ;免疫印迹分析表明 ,小鼠MAdCAM 1剪切体蛋白可被小鼠MAd CAM 1分子的标准单抗及自制多抗识别。结论 小鼠MAdCAM 1剪切体cDNA、表达蛋白及多抗的获得 ,为进一步研究该分子的生物学活性做好准备。

Objective To obtain the spliced mouse mucosal addressin cell adhesion molecule-1 (mMAdCAM-1) cDNA and express it in E.coli. Methods Obtaining the spliced mMAdCAM-1 cDNA from mesenteric lymph nodes of BALB/c mice by RT-PCR, cloning the cDNA into the procaryotic expressive vector pGEX-2T and expressing the fusion protein. Results After identified by DNA sequencing, the fusion protein was expressed and mainly existed as inclusion bodies. After purification, we prepared the multi clonal-antibody to the fusion protein. The spliced mMAdCAM-1 protein was identified by the multi clonal-antibody and standard monoclonal antibody against mMAdCAM-1 with Western blot. Conclusion The spliced mMAdCAM-1 cDNA, expressed protein and multi clonal-antibody can support the further research about the biological activities of mMAdCAM-1.