摘要
以PCR扩增猪带绦虫TS61抗原基因 ,与载体 pUC18连接后进行测序 ;并构建了该基因的pGEX 1λT原核表达载体 ,制备了其原核表达产物 ,进行浓度梯度SDS PAGE和免疫印迹分析 ;对该基因的碱基序列及其编码蛋白进行了同源性比较。结果表明 ,猪带绦虫TS61抗原基因含 893对碱基 ,编码含 70个氨基酸残基的多肽 ,分子质量为 8.0ku ,等电点为4.19。在基因库和欧洲分子生物学实验室数据库中均未发现该基因的同源序列。重组质粒在大肠埃希氏菌中表达的融合蛋白分子质量为 3 4ku ,该蛋白抗原能被兔抗猪囊尾蚴超免疫血清所识别。
To sequence,analyzeand express antigenic gene TS61 in Escherich coli, which was isolated from cDNA expression library of Cysticercus cellulosae, the TS61 cDNA fragment was amplified with PCR, and the product was digested with EcoRⅠ and ligated into pUC18, the recombinant was sequenced. The sequence was analyzed and compared with other genes of Taenia solium in GenBank and EMBL database. The fragment was ligated into expression vector pGEX 1λT, and then the recombinant plasmid that expressed the protein correctly was isolated with immunoscreening. The plasmid was used to transformed the DH5α of E.coli, and the transformed E.coli were induced to express the fusion protein by IPTG. The expression product was analyzed with SDS PAGE and Western blotting. The results indicated that the length of the TS61 gene is 893 bp, which encodes a predicted polypeptide of 70 amino acid residueswith molecular weight of 8.0 ku, pI 4.19, and there is no homologous sequence of TS61 gene in GenBank and EMBL database; the molecular weight of the fusion protein is 34 ku which was expressed with the recombinant pGEX 1λT plasmid in E.coli and gives weak positive signal in Western blotting with rabbit antisera against cysticercus, although it leads to strongpositive reaction in immunoscreening assay in situ, These demonstrated that a novel gene encoding immunogenicprotein of Cysticercus cellulosae is successfully isolatedin our experiment.
出处
《中国兽医科技》
CAS
CSCD
北大核心
2002年第5期13-17,共5页
Chinese Journal of Veterinary Science and Technology
基金
高等学校博士学科点专项科研基金资助项目 (941 0 0 6)
