鸡毒害艾美耳球虫MIC5基因在大肠杆菌的重组表达产物抗原性分析-预防兽医学专业论文.docxVIP

摘‘ 摘‘ 要 从鸡毒害艾美耳球虫孢子化卵囊中提取基因组DNA，用Oli906．0软件设计EnMIC．5基因特异 性引物，通过PCR扩增出l 470 bp的片段，扩增产物再连接到pMDl8．T载体中，经PCR扩增、酶 切鉴定和测序分析后证明所克隆的l 470 bp片段核苷酸序列及其编码的氨基酸序列与GenBank中登 录的微线蛋白5基因序列的相似性分别是99．7％和99．6％。 按照pET-28(a)+载体的特点和克隆位点，用Oli906．0软件设计了含BamH I和Xho I酶切位点的 EnMIC．5基因的特异性表达引物，以重组pMDl8．T-EnMIC．5质粒DNA为模板进行PCR扩增，扩 增产物经BamH I和Xho I双酶切后，将外源EnMIC．5基因亚克隆到pET-28(a)+表达载体中，构建 pET-EnMIC．5重组表达载体，转化大肠杆菌BL21(DE3)，经酶切、PCR和测序鉴定证明EnMIC．5 基因正确插入到pET-28(a)+载体中。经ITPG诱导表达，表达产物通过SDS和Western-blotting 分析证实EnMIC．5基因在BL21中成功表达，分子质量大小为57．5 lcu左右，与预期的分子质量大小 一致，且能被鸡毒害艾美耳球虫阳性血清所识别；将重组蛋白免疫昆明小鼠，用间接ELISA检测， 证实该重组蛋白具有良好的免疫原性，可诱导小鼠产生高滴度特异性抗体。 本研究为鸡球虫微线蛋白结构与功能研究提供了依据和实验材料，为进一步研制基因工程疫苗 提供了候选抗原。 关键词：毒害艾美耳球虫，微线蛋白5，基因克隆，重组表达，免疫原性 AbstractGenomic Abstract Genomic DNA was extracted from the oocyst of Eimeria necatrix,and the EnMIC-5 gone specific primers were designed USillg Oli906．0 sottware．A 1 470 bp specific fragment was amplified by PCR and ligated into pMDl 8·T vector．The recombinant plasmid was identified by PC氏restriction endonuclease analysis and sequencing．The similarity ofthe nucleotide sequence and deduced amino acid sequence ofthe EnMIC·5 gene was 99．7％and 99．6％．respectively compared with the published sequence in GenBank． According to the characterization and the cloning site of pET-28(a)+vector,the specific primers were designed．Then the fragment Was subcloned into the BamH I and Xho I sites of the prokaryotic expression vector pET-28(a)+and Was transformed into E coli BL21(DE3)．Expression of the recombinant plasmid Was induced by IPTG in E coli,and the expressed products were analyzed by SDS—PAGE and Western-blotting．The results of SDSshowed that the fusion protein with molecular weight of about 57．5 ldga Was over-expressed．Western-blotting demonstrated that the expressed recombinant protein Was reacted with anti E necatrix positive sera from chickens．The Kunming mice were immunized with the purified recombinant protein and their were examined by ELISA．The results suggested that the recombinant pro