UGRP1蛋白的表达、纯化、抗体制备及亚细胞定位

Cloning,expression and polyclonal antibody preparation of UGRP1 and its subcellular localization

目的子宫球蛋白相关蛋白1(UGRP1)是一种功能未知的分泌蛋白。通过表达、纯化蛋白、制备抗体及其在组织中表达的研究,为进一步研究其功能奠定基础。方法从人胎肺组织中克隆得到UGRP1基因,构建重组表达质粒pGEX-5X-1/UGRP1。将此质粒转化大肠杆菌BL21(DE3)后用异丙基-β-D-硫代半乳糖苷(IPTG)诱导,表达含谷胱甘肽-S-转移酶(GST)的GST-UGRP1融合蛋白。此融合蛋白经纯化分离后,免疫新西兰大白兔,制备其多克隆抗体。用Western印迹的方法检测多抗血清。通过构建含增强型绿色荧光蛋白(EGFP)的EGFP-N2-UGRP1载体,转染非洲绿猴肾细胞(COS细胞)表达重组蛋白,对UGRP1的表达进行亚细胞定位。结果成功构建了pGEX-5X-1/UGRP1载体,获得了高纯度的重组蛋白和多克隆抗体。免疫组化分析表明UGRP1表达于肺支气管上皮细胞,亚细胞定位分析证实UGRP1主要表达于细胞浆。结论研究发现UGRP1表达于肺支气管上皮细胞和在COS细胞的胞浆中表达。经纯化获得蛋白并制备抗体,为进一步研究UGRP1的功能奠定了基础。

To prepare uteroglobin-related protein 1 （UGRP1） fusion protein, an unknown secretion protein, and its polyclonal antibody for following function study. Methods The coding region of UGRP1 was amplified from a human embryo lung tissue by RT-PCR and the recombinant prokaryotic expression vector pGEX-UGRP1 was constructed. The vector was transformed into E. coli BL21 （DE3） to expresse the glutathione-S-transferase （GST）-UGRP1 fusion protein in the bacteria under induction of isopropy113-D-l-thiogalactopyranoside （IPTG）. After purification, the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by Western Blotting for their sensitivity and specificity. The full sequence of UGRP1 gene was also amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the EGFP-N2 vector. The recombined vector EGFP-N2-UGRP1 was transfected into COS cells to investigate its expression with laser scanning confocal microscope. Results The recombinant prokaryotic expression vector pGEX-UGRP1 was constructed successfully, then GST-UGRP1 fusion protein and its polyclonal antibody were also generated. Immunohistochemical assay, by using this antibody, demonstrated that UGRP1 had high expression in the epithelial cells of the airways, while it＇s confirmed that UGRP1 was predominant expression in the cytoplasm. Conclusion Preparation of UGRP1 fusion protein and its polyclonal antibody,together with preliminary study of UGRP1 expression characteristics, may lay foundation to investigating the function of UGRP1 gene in the future.