MUC1/Y特异表位肽在大肠杆菌中的可溶性表达及其抗体的制备  

Soluble Expression of Peptide Containing MUC1/Y-specific Epitope in Escherichia coli and Preparation of the Antibody

作　　者：张立新[1] 李春海[1] 孙丽亚[1] 王淼[1] 路浩军[1] 

机构地区：[1]军事医学科学院附属医院肿瘤分子生物室,北京100039

出　　处：《生物工程学报》2003年第3期337-342,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金重点课题资助 (No .3 983 0 3 3 0 );863课题资助 (No .2 0 0 2AA2 14 111)~~

摘　　要：为获得MUC1 Y的特异表位肽 ,制备抗体 ,用PCR扩增其编码序列 ,克隆到pGEX 2T中 ,转化DH5α感受态 ,0 2mmol LIPTG诱导表达 ,超声破碎或B PERTMⅡ试剂处理诱导菌 ,亲和层析和阴离子交换纯化目的蛋白 ,SDS PAGE及Westernblotting鉴定 ;免疫家兔制备多抗 ,初步用于免疫组化分析。结果表明 ,转化菌经诱导后表达融合蛋白GST Y30 ,约占菌体总蛋白的 2 0 % ,大多以可溶形式存在 ,与诱导温度无关。免疫家兔获得多抗 ,效价为1∶32 0 0 0 0 ,纯化的抗体具有特异性 ,可识别肿瘤细胞表面的MUC1 Y蛋白。所得蛋白和抗体可用于MUC1 Y的表达特征及其生物学功能研究。MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y specific epitope in E. coli and prepare MUC1/Y specific antibody, DNA fragment encoding the MUC1/Y specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX 2T, resulting pGEX Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5α was transformed with pGEX Y30 and the expression was induced for 4～5 hours in 0 2mmol/L IPTG at 30℃ and 37℃. Expressed proteins were released from the cells by ultrasonication or B PER TM Ⅱ reagent treatments. The fusion protein GST Y30 were purified by affinity and anion exchange columns and identified by SDS PAGE and Western blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y binding capacity and specificity. The expressed fusion protein GST Y30 is about 31kD in size and represented about 20% of total cellular proteins. The majority of the GST Y30 protein existed as soluble form when the induction was carried out at both 30℃ and 37℃. After the two step purification, the purity of GST Y30 was about 94%. The titer of polyserum generated by GST Y30 immunization was 1∶320 000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y sp

关 键 词：肽 纯化 抗体 大肠杆菌 可溶性表达 

分 类 号：Q789[生物学—分子生物学]