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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。
Objective To determine and analyze the sequence of the cDNA clone (TS76) screened from the cDNA library of Cysticercus cellulosae and to study the prokaryotic expression of the fragment. Methods The phage (TS76 cDNA fragment in TS76, subcloned into pUC18) was amplified by PCR and sequenced. The results were analyzed and compared. The TS76 fragment was subcloned into prokaryotic expression vector pGEX - 1 (T, the recombinant plasmid pGTS76 was successfully screened by Western blotting, and the prokaryotic expression product of pGTS76 was prepared and analyzed by concentration gradient SDS-PAGE and Western blotting.RESULTS The TS76 clone contained 5 16 pairs of nucleotides, 83 amino acid residues of the polypeptide, and EMBL database of the cysticerci immunogenic protein mRNA 383bp homologous regions of the recombinant plasmid expressed in E. coli fusion protein molecular weight of 34KD, anti-Cysticercus cellulosae rabbit serum Produced a strong immune response.ConclusionA gene encoding a highly immunogenic cysticercus antigen was isolated.