【作者】 王喆；

【导师】 崔玉东；

【作者基本信息】 黑龙江八一农垦大学 ， 预防兽医学， 2010， 硕士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus, S. aureus)是引起人类皮肤、粘膜感染和动物多种疾病的重要致病菌。由于不同S. aureus菌株间交叉免疫保护性有限,目前研究的S. aureus疫苗在临床试验中效果不理想。S. aureus的铁元素获取体系(Isd)对细菌的生存至关重要,IsdA是该系统的重要组成部分。因此,本试验对S. aureus表面蛋白IsdA进行原核表达,探讨其免疫保护作用。实验首先对S. aureus Newman株的isdA基因进行PCR扩增,并连接pMD-18T载体进行测序,测序结果与GenBank已发表的基因序列进行比较。构建pET32a-isdA原核表达载体,转化到Rosetta(DE3)中,经IPTG诱导后,用SDS-PAGE鉴定IsdA,并将重组蛋白与S. aureus全菌体抗血清进行Western Blot,结果证明重组IsdA蛋白具有免疫原性。与此同时,对重组IsdB蛋白进行诱导表达及纯化,用以对比IsdA蛋白的免疫保护效果。取120只健康小鼠随机分成12小组,每3小组分别组成IsdA蛋白免疫组、IsdB蛋白免疫组、IsdA+IsdB蛋白免疫组、无免疫原对照组,各组蛋白分别与弗氏完全佐剂、弗氏不完全佐剂充分乳化制备免疫原。另取12只小鼠随机分成4组,按上述方法分别免疫,用以跟踪抗体效价。初次免疫后,每隔一周对跟踪抗体效价组小鼠采血,应用ELISA方法检测血清中IgG抗体水平,并在第一次免疫后第一周检测血清中IFN-γ、IL-4细胞因子浓度。初免三周进行第二次免疫,于第二次免疫后第二周分别用S. aureus Wood46、Newman和HLJ855-23-1株对每组免疫小鼠攻毒,连续观察死亡情况并记录一周。小鼠免疫试验结果表明,表达的重组蛋白IsdA+IsdB蛋白联合免疫具有较好的免疫保护作用。通过对细胞因子IFN-γ和IL-4的细胞免疫检测,和IgG抗体水平的检测,结果表明IsdA+IsdB蛋白联合免疫组可有效刺激小鼠模型的细胞免疫应答和体液免疫应答,应答水平明显高于对照组和蛋白单独免疫组。更多还原

【Abstract】 Staphylococcus aureus (S. aureus) is an important human and animal pathogen. S. aureus is found on the skin and mucosal surfaces of humans and cause a variety of important diseases of animal. As cross-protective immunity of different S. aureus strains is limited, the present study of S. aureus vaccine in clinical are not ideal. S. aureus iron-regulated surface determinant (Isd) is essential for the survival of bacteria, and IsdA play a key role in this system. IsdA, the surface protein of S. aureus, was prokaryotic expressed and prepared immunogen, to explore the protective effects of immunization of IsdA.Firstly, isdA gene of S. aureus strain Newman was amplified by PCR, and was ligated into pMD-18T vector and sequenced, sequencing results compared with the one published previously in GenBank. Construction of the pET32a-isdA prokaryotic expression vector, it was expressed in Rosetta (DE3) after induced with IPTG. The recombinant IsdA fusion protein was detected by SDS-PAGE. Western blot assay were performed to characterize the immunogenicity of the recombinant IsdA protein. At the same time, the recombinant IsdB protein was expressed and purified, to compare the protective effects of immunization of IsdA protein. Then, 120 healthy mice were divided into 12 groups for the IsdA protein group, IsdB protein group, IsdA + IsdB protein group, non-immunized control group. Each group protein was emulsified with Freund’s complete adjuvant and Freund’s incomplete adjuvant for preparation of immunogen. Other twelve healthy mice were divided into 4 groups, immunized by the same method to measure the antibody titer. Every group for getting serum was taken serum every week and IgG antibody levels in serum were detected by ELISA. IFN-γ, IL-4 cytokines levels in serum that collected in the first week after the frist immunization were detected by ELISA. The second immunization begins the third week after the first immunization, and challenged by S. aureus strains Wood46, Newman, HLJ855-23-1 in the second week after the second immunization, record the death for one week.Immunity test results show that the expressed recombinant protein IsdA + IsdB combined immunization have a preferable immunoprotection. By the detection of cell immunity with cytokines IFN-γand IL-4, and the IgG antibody levels, the test results show that the IsdA + IsdB combined immunization can effectively stimulate cellullar immunologic response and humoral immunoresponse in mice, and the response level is significantly higher than the control group and the protein immunization alone.更多还原