【作者】 赵艳；

【导师】 郝永清；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 金黄色葡萄球菌是引起奶牛乳腺炎的一种主要传染性病原菌。本实验运用了分子克隆技术及蛋白表达技术,成功的表达了金黄色葡萄球菌纤维素结合蛋白A(FnBPA)D区的蛋白并对其免疫学活性进行了研究。本研究根据GenBank上公布的金黄色葡萄球菌FnBPA D区的序列,设计了特异性引物,提取分离自内蒙古地区的金黄色葡萄球菌基因11型和基因3型的基因组DNA后进行PCR,并与T载体连接进行克隆。克隆产物经核酸序列测定后表明已成功克隆了目的基因。测序后分别与金黄色色葡萄球菌NCTC 8325株的FnBPA进行比对发现,基因11型和基因3型与其核苷酸同源性分别为95%和94%。基因3型与基因11型之间核苷酸的同源性为96.5%。用DNAstar对测序片段进行抗原位点分析,结果显示,基因11型要比基因3型和标准菌株多一个抗原位点,由此决定表达基因11型的FnBPA D区蛋白。将目的片段与表达载体pET-32(a)连接,连接产物转化宿主菌BL21(DE3)后,用IPTG进行诱导,确定最佳诱导时间,大量表达并纯化出了目的蛋白。表达产物经两次免疫实验动物后,获得较高效价的抗体。用自制血清进行吞噬调理试验,抗金黄色葡萄球菌黏附等一系列试验,证明抗体对金黄色葡萄球菌具有吞噬调理作用,也有抗金黄色葡萄球菌黏附的作用,这将为制备奶牛乳房炎金黄色葡萄球菌黏附素疫苗奠定基础。更多还原

【Abstract】 The staphylococcus aureus is a kind of main contagion cause germ which causes milk cow mastitis.This study using molecular cloning technique and protein expression technique expresses region D of fibronectin-binding protein A gene of Staphylococcus aureus successfully and research its immunologic competence.Region D of fibronectin-binding protein A gene (FnBPA gene)of Staphylococcus aureus is amplified with PCR method, on the basis a pair of specific primers designed according to the relevant nucleotide sequence from GeneBank. We use genome DNA of gene type 3 and gene 11 which separated from Inner Mongolia .After PCR, we complete cloning of FnBPA D region. The positive recombinant clones was sequenced and analysed. The results suggested that FnBPA D region gene was cloned successfully. Then we blast the result with relevant nucleotide sequence on GeneBank. The result shows that the autoploidies of gene type 3 and gene 11 with the sequence on GeneBank are 98% and 94%. And the autoploidy of gene type 3 and gene 11 with is 96.5%.Antigen sites are analysised by the software DNAstar, the result shows gene type 3 has more antigen site than the other two sequences. So we decide to express the gene type 3. The fragment is successfully cloned into pET-32a(+)to construct a recombinant expressed vector pET- FnBPA. The recombinant expressed plasmid is transformed into the host cell E.coli BL21(DE3)and induced by IPTG and then depurated the protein.After experimental animals are immuned by expression product two times, we obtain high potency antibody. Then carry out regulation and anti-binding of staphylococcus aureus experiments, and the results show that the anbody has the contribution of immunoprotection and anti-binding of staphylococcus aureus. This can play a role in research pathogenesis of staphylococcus aureus and preparing vaccine for mastitis.更多还原