摘要
目的:构建钙整合素结合蛋白(calcium-and inte-grin-binding protein,CIB)的原核表达载体,制备小鼠抗人CIB的多克隆抗体并探讨其亚细胞定位。方法:采用RT-PCR方法从人脑组织扩增CIB的cDNA片段,利用DNA重组技术构建原核表达载体pET32a-CIB,转入BL21(DE3)中,以IPTG诱导BL21(DE3)表达CIB融合蛋白,SDS-PAGE鉴定融合蛋白CIB的表达。用含CIB融合蛋白的聚丙烯酰胺凝胶颗粒为抗原免疫小鼠制备抗血清,抗血清分别经Protein G、抗原亲和层析后用Western blot及免疫组化方法进行鉴定。结果:通过重组表达获得CIB抗原蛋白,免疫小鼠并纯化抗血清得到特异性的抗CIB抗体,该抗体能检测细胞内源性CIB蛋白的表达;同时免疫组化结果显示:CIB在SHG44、Hhu7细胞株中主要定位于细胞质。结论:成功克隆CIB基因并制备了特异性的小鼠抗人的CIB多克隆抗体,对进一步研究CIB的功能奠定了基础。
AIM： To construct the prokaryotic expression vector pET32a-CIB, prepare the specific polyclonal antibody against CIB and study the subcellular localization of CIB. METHODS： CIB was amplified by RT-PCR from human brain tissue and cloned into prokaryotic expression vector pET32a-CIB. The CIB fusion protein was expressed in BL21 （DE3）/pET system and identified by SDS-PAGE. The mice were immunized with the polyacrylamide gel particles containing the CIB fusion protein for polyclonal antibody preparation. The antibody was purified by affinity chromatographic column matrix coupled with protein G, antigen respectively and then identified by Western blot and immunohistochemistry. RESULTS： The protein of CIB was obtained by recombination expression. The specificity of polyclonal antibody was obtained by immunizing BALB/c mice with polyacrylamide gel particles containing the fusion protein of CIB and purification. The results of immunohistochemistry demonstrated that CIB was localized predominantly in the cytoplasm of SHG44 and Hhu7 cells. CONCLUSION： The protein of CIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of CIB has been obtained, which can be used for further research into the function of CIB.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第1期58-61,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30400125)
中国博士后科学基金资助项目(20070410230)
关键词
CIB
基因重组
融合表达
多克隆抗体
亚细胞定位
CIB
gene recombination
fusion expression
polyclonal antibody
subcellular localization
