肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定

Molecular Cloning of MUC1/Y and Soluble Expression of Its Extra-cellular Fragment in E.coli

为获得MUC1 Y全长cDNA及其胞外段蛋白 ,以用于进一步的生物学功能及肿瘤生物学治疗的研究 .利用RT PCR从HeLa细胞中扩增MUC1 Y全长cDNA ;PCR扩增其胞外段 ,克隆到原核表达载体pGEX 2T ,转化DH5a菌 ,诱导表达 ;亲和层析纯化 ;凝血酶酶切、GST活性及N端蛋白测序鉴定 ;免疫家兔制备多克隆抗体 .所得MUC1 YcDNA的开放读框为 759bp ,登录于GenBank(AF12 552 5) .其信号肽编码序列缺失 9bp ,第 3 3 1位发生G A转换 ,造成缬氨酸突变为蛋氨酸 .表达获得约 4 0kD融合蛋白GST Yex ,占菌体总蛋白 2 5%～ 3 0 % ,其中 70 %～ 80 %为可溶性 ,经亲和层析一步纯化 ,纯度 >90 % ,GST比活性为 0 2 1U μg .凝血酶酶切后的N端蛋白序列测定表明与已知序列完全一致 ,抗血清ELISA效价为 1∶2 50 0 0 0 .结果表明 ,克隆到发生碱基缺失和突变的MUC1 Y全长cDNA ,获得MUC1 Y胞外段蛋白及其多抗 ,可进一步用于相关研究 .

To clone MUC1/Y full length cDNA and express and purify its extra\|cellular fragment protein from E.coli for further functional and tumor therapeutic research purposes.MUC1/Y cDNA was amplified by RT\|PCR from HeLa cells and cloned into pGEM\|T for sequence analysis.MUC1/Y extra\|celluar fragment (MUC1/Yex) was then amplified by PCR and cloned into pGEX\|2T fusion expression vector. E.coli DH5α was transformed with the new constructed expression vector pGEX\|Yex and induced by IPTG.The fusion protein GST\|Yex was purified by affinity column and identified by thrombin digestion,GST activity and N\|terminal protein sequencing.Polyclonal antibody was prepared by immunizing rabbits.The results showed that the open reading frame(ORF) of MUC1/Y cDNA from HeLa cells consisted of 759 bp with a 9 bp deletion in its signal peptide\|coding region and a G to A transition at the site of 331 bp which caused a V to M amino acid mutation (GenBank access number AF125525).The expressed fusion protein GST\|Tex was about 40 kD which was 25%-30% of total host proteins.70%-80% of the GST\|Yex proteins existed as soluble ones and after one\|step affinity purification the purity of GST\|Yex was over 90% with 0\^21 U/μg GST activity.MUC1/Yex protein can be cleaved from GST\|Yex and N\|terminal protein sequencing confirmed that is was consistent with the sequence published.The titer of polyserum generated was 1∶250 000 by ELISA.In conclusion,MUC1/Y full length cDNA was cloned.MUC1/Yex protein and its polyclonal antibody were obtained for further research purposes.