%0 Journal Article
%T Soluble Expression of Peptide Containing MUC1/Y-specific Epitope in Escherichia coli and Preparation of the Antibody<br>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
%A ZHANG Li Xin
%A LI Chun Hai SUN Li Ya WANG Miao LU Hao Jun
%A <br>张立新
%A 李春海
%A 孙丽亚
%A 王淼
%A 路浩军
%J 生物工程学报
%D 2003
%I 
%X 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 DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER 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 31 kD 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 degrees C and 37 degrees C. 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-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.
%K MUC1/Y
%K peptide
%K expression
%K purification
%K antibody<br>MUC1/Y，
%K 肽，
%K 表达，
%K 纯化，
%K 抗体
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=3CA917D208C35846&yid=D43C4A19B2EE3C0A&vid=2A8D03AD8076A2E3&iid=38B194292C032A66&sid=5FF9F4F7CB1800C7&eid=556C1A86E372B606&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=1&reference_num=18