APECO78前噬菌体蛋白的重组表达及血清制备
时间:2018-09-20 16:28 来源:毕业论文 作者:毕业论文 点击:次
| 摘要：通过比较基因组学分析得出，禽致病性大肠杆菌O78血清型的菌株中存在两个特异性基因-噬菌体溶菌酶基因 AGC87669.1和整合酶基因AGC87680.1。用PCR方法对这两个基因在实验室保存的144株大肠杆菌中的分布做了调查，结果显示这两个基因确实只在O78血清型菌株中检测为阳性，其他菌株中检测结果均为阴性。对这两基因进行了克隆表达，用PCR方法扩增出AGC87669.1、AGC87680.1基因后，分别连接到表达载体pET-32a中，并转化入DH5ɑ中，提取重组质粒再转化入BL21中制备重组表达菌BL21-pET-32a-AGC87669.1，BL21-pET-32a-AGC87680.1，并用IPTG诱导表达。诱导产物通过SDS-PAGE检测，在40.44kDa 、62kDa处出现与预期相一致的蛋白条带。将这两个蛋白进行了纯化，并制备了免疫血清，为后续蛋白免疫原性和保护性抗原的研究打下基础。28394 毕业论文关键词：APEC O78；前噬菌体蛋白；克隆表达；融合蛋白的表达；融合蛋白的纯化 Immunogenicity analysis of prophage protein in APEC O78 Abstract：Through comparative genomics analysis, there were two specific gene lysozyme gene AGC87669.1 and integrase gene .AGC87680.1 in the strains of avian pathogenic Escherichia coli O78.The distribution of the two genes in 144 strains of Escherichia coli stored in the laboratory was tested by PCR, and the results showed that the two genes were positive only in O78 strains, and the results were negative in other strains. Cloning and expression of the two genes, the AGC87669.1 and AGC87680.1 genes were amplified by PCR, respectively connected to the expression vector pET-32a and transformed into the DH5ɑ, the recombinant plasmid was transformed into the BL21 and prepared recombinant expressing bacteria BL21-pET-32a-AGC87669.1 and BL21-pET-32a-AGC87680.1,and expressed by IPTG induction. The induced products were detected by SDS-PAGE, and the stripe 40.44 kDa and 62 kDa are consistent with expectation. The two proteins were purified and the immune serum was prepared, which laid the foundation for the study of the immunogenicity and protective antigen of the protein. Key words: APEC O78；prophage protein；expression of fusion protein；purification of fusion protein 摘要 1 关键词 1 Abstract 1 Key words 1 引言 1 1 材料与方法 2 1．1 实验材料 2 1．1．1 大肠杆菌菌株 2 1．1．2 质粒及感受态细胞 3 1．1．3 试剂 3 1．1．4 仪器 3 1．1．5 试剂配方 4 1．2 方法 4 1．2．1 AGC87669.1、AGC87680.1基因在大肠杆菌中国的分布调查 4 1．2．2 AGC87669.1、AGC87680.1蛋白的重组表达载体的构建 5 1．2．3 融合蛋白的表达 6 1．2．4 融合蛋白的纯化 6 1．2．5 兔抗血清的制备 7 2 结果与分析 7 2．1 AGC87669.1、AGC87680.1基因在大肠杆菌中国的分布 7 2．2 重组表达载体的构建与鉴定 9 2．3 融合蛋白的表达与纯化 9 3 讨论 10 致谢 11 参考文献： 11 APEC O78前噬菌体蛋白的重组表达及血清制备 大肠杆菌是大肠埃希氏菌（Escherichia coli, E coli）的俗称，属肠杆菌科、埃希氏菌属，1885年由德国细菌学家Theodor Escherich自婴儿粪便中分离出来，因此命名为大肠埃希氏菌[1]。禽致病性大肠杆菌(avian pathogenic Escherichia. coli, APEC)可引起禽类的脐炎、腹膜炎、心包炎、气囊炎、肝周炎、输卵管炎、滑膜炎以及败血症等多种疾病, 目前严重危害养禽业[2, 3]。 (责任编辑：qin) |