猪囊尾蚴抗原基因Ts11的克隆及其在大肠杆菌的表达

CLONING AND EXPRESSION OF ANTIGENIC GENE Tsll OF CYSTICERCUS CELLULOSAE

PCR扩增重组噬菌体λTs11克隆的cDNA片段 ,与pUC18连接 ,得到的重组子用以测序 ,并进行同源性比较分析 ;将该片段克隆到原核表达载体pGEX 1λT中 ,免疫印迹筛选得到能正确表达该片段的重组子 ;制备该重组子在大肠杆菌中的表达产物 ,进行浓度梯度SDS PAGE和West ernblotting分析。结果表明Ts11cDNA片段为 52 2bp ,编码含 139个氨基酸残基的多肽 ,在Gen Bank和EMBL数据库均未发现同源序列。重组质粒在大肠杆菌中表达的融合蛋白分子量为 4 1KD ,能与兔抗猪囊尾蚴囊液血清产生很强的免疫反应。这些结果证实分离到一个新的编码具较强免疫原性猪囊尾蚴抗原的cDNA克隆。

The cDNA fragment was amplified using PCR,and the product was digested and ligated into pUC18,the recombinant plasmid was sequenced.The sequence was analyzed and compared in GenBank and EMBL database.Then the fragment was ligated into expression vector pGEX 1λT,the recombinant plasmid,which could express the protein correctly was isolated by immunoscreening.The recombinant then was cultured,and the fusion protein was induced to express by IPTG.The expressed product was analyzed by SDS PAGE and Western blotting.The results show that the length of this cDNA clone is 522 bp,which encodes a predicted polypeptide of 139 amino acid residues.There is no homologous sequence in GenBank and EMBL database.The molecular weight of the fusion protein,which was expressed by recombinant plasmid in E.coli,is 42KD,the fusion protein gives strong positive signal after Western blotting with rabbit anti cysticercus sera.A novel cDNA clone encoding immunogenic antigen of Cysticercus cellulosae was isolated.