猪带绦虫抗原基因TS76的克隆及原核表达

目的　测定和分析自猪囊尾蚴表达型cDNA文库中筛选出的cDNA克隆 (TS76 )的序列 ,并进行该片段的原核表达研究。方法　采用PCR扩增重组 (噬菌体 (TS76中的TS76cDNA片段 ,亚克隆至 pUC18,得到的重组子用于测序 ,并对测定结果进行分析及同源性比较 ;将TS76片段亚克隆到原核表达载体pGEX - 1(T中 ,免疫印迹方法筛选得到能正确表达TS76片段的重组子 pGTS76 ;制备pGTS76原核表达产物 ,进行浓度梯度SDS -PAGE和Westernblotting分析。 结果　TS76克隆含 5 16对核苷酸 ,编码含 83个氨基酸残基的多肽 ,与EMBL数据库中的猪囊虫免疫原性蛋白质mRNA有 383bp的同源区域。重组质粒在大肠杆菌中表达的融合蛋白分子量为 34KD ,能与抗猪囊尾蚴兔血清产生很强的免疫反应。结论　分离到一个编码具较强免疫原性猪囊尾蚴抗原的基因。

Cloning and prokaryotic expression of antigenic gene of TS76 of Taenia solium

Aim To analyze the sequence of the TS76 cDNA clone isolated from cDNA expression library of Taenia solium and express it in E coli Method The cDNA fragment was amplified using PCR,and the product was digested and subcloned into pUC18,the recombinant was sequenced The sequence of TS76 was analyzed and compared with other genes of Taenia solium in GenBank and EMBL database Then the fragment was ligated into expression vector pGEX 1λT,the recombinant plasmid expressing the protein correctly was isolated by immunoscreening The plasmid was then cultured and was induced to express the fusion protein by IPTG The expression product was analyzed by SDS PAGE and Western blotting Results The length of this cDNA clone is 516bp,which encodes a predicted polypeptide of 83 amino acid residues It has a 383bp homologous region with the Taenia solium mRNA for immunogenic protein in EMBL database The molecular weight of the fusion protein is 34KD which was expressed by recombinant plasmid in E coli,and it gives strong positive signal after Western blotting with rabbit antisera against cysticercus Conclusion A cDNA clone encoding immunogenic protein of Taenia solium cysticercus was successfully isolated