贝氏柯克斯体外膜蛋白与热休克蛋白融合蛋白的研制及其免疫原性的研究
发布时间：2018-11-12 21:08
【摘要】： 贝氏柯克斯体是一种专性细胞内寄生菌，为重要人畜共患病Q热的病原体，也是一种重要的生物战剂和生物恐怖剂。Q热疫苗接种是预防贝氏柯克斯体感染的最有效的措施。采用基因工程技术制备重组保护性抗原是研制Q热分子疫苗的重要手段。 贝氏柯克斯体的外膜蛋白P1(分子量为29kDa)、Com1(分子量为27kDa)以及34kDa蛋白(SP34)是重要的保护性抗原，热休克蛋白B(HspB)也是其主要表面抗原。另外许多研究证明热休克蛋白具有的佐剂样功能，能增强其它抗原的免疫保护效能。因此，本研究研制了贝氏柯克斯体HspB融合于P1、Com1、SP34外膜蛋白，制备了热休克蛋白融合蛋白，并对这些融合蛋白的免疫原性和免疫保护性进行评价。 采用分子克隆技术，从贝氏柯克斯体基因组中克隆P1、Com1、SP34外膜蛋白基因以及HspB基因；将外膜蛋白基因分别与HspB基因融合构建了pQE30／p1-htpB、pQE30／com1-htpB、pET28a／htpB-sp34原核表达质粒，原核表达质粒转化的大肠杆菌分别产生P1-HspB、Com1-HspB、HspB-SP34融合蛋白。免疫印迹分析显示这些融合蛋白均可以与贝氏柯克斯体感染血清发生特异性的反应，证明其具有良好的抗原反应性。 采用亲和层析的方法纯化P1-HspB、Com1-HspB、HspB-SP34融合蛋白，用它们分别免疫BALB／c小鼠，分析其免疫原性。P1-HspB融合蛋白免疫小鼠不仅产生特异性IgG抗体的水平显著高于单P1蛋白免疫小鼠，而且γ干扰素和IL-2细胞因子水平也显著高于P1单个蛋白免疫。检测Com1-HspB和HspB-SP34融合蛋白免疫小鼠后的抗体水平，结果同样表明融合蛋白诱导机体产生特异性抗体的能力显著好于外膜蛋白单独免疫。 将纯化的P1-HspB、Com1-HspB、HspB-SP34融合蛋白分别免疫BALB／c小鼠，用贝氏柯克斯体毒株对免疫小鼠进行攻击，最后用定量PCR分析受攻击小鼠脾脏的贝氏柯克斯体载量。多次试验的结果显示融合蛋白免疫小鼠的贝氏柯克斯体攻击后脾脏荷菌量显著低于单独外膜蛋白免疫小鼠，说明热休克融合蛋白P1-HspB、Com1-HspB、HspB-SP34的免疫保护效果明显好于P1、Com1、SP34、HspB单独免疫。 结论：贝氏柯克斯体膜表面蛋白与热休克蛋白B融合后产生的P1-HspB、Com1-HspB、HspB-SP34融合蛋白的免疫原性和免疫保护性显著强于单个外膜蛋白，，说明热休克蛋白B具有显著的免疫增强作用。P1-HspB、Com1-HspB、HspB-SP34融合蛋白可作为研制Q热分子疫苗的候选分子。
[Abstract]:Coxella Baysoni is a specific intracellular parasite, which is the pathogen of important zoonotic Q fever, as well as an important biological warfare agent and bioterrorist agent. Q fever vaccination is the most effective measure to prevent the infection of Coxella bassii. The preparation of recombinant protective antigen by genetic engineering is an important method to develop Q-Fever molecular vaccine. The outer membrane proteins P1 (molecular weight of 29kDa), Com1 (molecular weight of 27kDa) and 34kDa protein (SP34) are important protective antigens, and the heat shock protein B (HspB) is also the main surface antigen. Many other studies have demonstrated that heat shock proteins have adjuvant-like functions that enhance the immune protection of other antigens. Therefore, in this study, the fusion protein of Coxella basicolor HspB into P1C 1 / SP34 outer membrane protein was developed, and the fusion proteins of heat shock protein were prepared, and the immunogenicity and immunological protection of these fusion proteins were evaluated. The outer membrane protein gene and HspB gene were cloned from the genome of Coxella basi by molecular cloning technique. The prokaryotic expression plasmids of pQE30/p1-htpB,pQE30/com1-htpB,pET28a/htpB-sp34 were constructed by fusion of outer membrane protein gene and HspB gene, respectively. E. coli transformed by prokaryotic expression plasmid produced fusion protein of P1-HspBnCom1-HspBN-HspB-SP34, respectively. The immunoblotting analysis showed that these fusion proteins could react specifically with the sera infected with Coxella bassii, which proved that these fusion proteins had good antigenic reactivity. The fusion protein of P1-HspBCon Com1-HspB-SP34 was purified by affinity chromatography. BALB/c mice were immunized with P1-HspBC-SP34 fusion protein. The immunogenicity of P1-HspB fusion protein immunized mice was significantly higher than that of single P1 protein immunized mice, and the levels of interferon 纬 and IL-2 cytokines were significantly higher than that of P1 single protein immunized mice. The antibody level of mice immunized with Com1-HspB and HspB-SP34 fusion protein was determined. The results also showed that the fusion protein could induce specific antibody in mice significantly better than that of outer membrane protein alone. BALB/c mice were immunized with purified fusion protein P1-HspBU Com1-HspBC-HspB-SP34. The mice were immunized with the strain of Coxella bassii. Finally, the load of Cocks' body in the spleen of the attacked mice was analyzed by quantitative PCR. The results of many experiments showed that the amount of splenic inoculation of mice immunized with the fusion protein was significantly lower than that of mice immunized with outer membrane protein alone, indicating that the heat shock fusion protein P1-HspBmember Com1-HspB was significantly lower than that of mice immunized with outer membrane protein alone. The immune protective effect of HspB-SP34 was better than that of P _ (1) C _ (1) P _ (34) HspB alone. Conclusion: the immunogenicity and immunological protection of P1-HspBU Com1-HspB-SP34 fusion protein produced by fusion of membrane surface protein of Cox's body and heat shock protein B are significantly stronger than that of single outer membrane protein. The results indicated that heat shock protein B (HSPB) had a significant immune enhancement effect, and the fusion protein P1-HspBU Com1-HspBC1-HspB-SP34 could be used as a candidate for the development of Q fever molecular vaccine.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378
本文编号：2328222
[Abstract]:Coxella Baysoni is a specific intracellular parasite, which is the pathogen of important zoonotic Q fever, as well as an important biological warfare agent and bioterrorist agent. Q fever vaccination is the most effective measure to prevent the infection of Coxella bassii. The preparation of recombinant protective antigen by genetic engineering is an important method to develop Q-Fever molecular vaccine. The outer membrane proteins P1 (molecular weight of 29kDa), Com1 (molecular weight of 27kDa) and 34kDa protein (SP34) are important protective antigens, and the heat shock protein B (HspB) is also the main surface antigen. Many other studies have demonstrated that heat shock proteins have adjuvant-like functions that enhance the immune protection of other antigens. Therefore, in this study, the fusion protein of Coxella basicolor HspB into P1C 1 / SP34 outer membrane protein was developed, and the fusion proteins of heat shock protein were prepared, and the immunogenicity and immunological protection of these fusion proteins were evaluated. The outer membrane protein gene and HspB gene were cloned from the genome of Coxella basi by molecular cloning technique. The prokaryotic expression plasmids of pQE30/p1-htpB,pQE30/com1-htpB,pET28a/htpB-sp34 were constructed by fusion of outer membrane protein gene and HspB gene, respectively. E. coli transformed by prokaryotic expression plasmid produced fusion protein of P1-HspBnCom1-HspBN-HspB-SP34, respectively. The immunoblotting analysis showed that these fusion proteins could react specifically with the sera infected with Coxella bassii, which proved that these fusion proteins had good antigenic reactivity. The fusion protein of P1-HspBCon Com1-HspB-SP34 was purified by affinity chromatography. BALB/c mice were immunized with P1-HspBC-SP34 fusion protein. The immunogenicity of P1-HspB fusion protein immunized mice was significantly higher than that of single P1 protein immunized mice, and the levels of interferon 纬 and IL-2 cytokines were significantly higher than that of P1 single protein immunized mice. The antibody level of mice immunized with Com1-HspB and HspB-SP34 fusion protein was determined. The results also showed that the fusion protein could induce specific antibody in mice significantly better than that of outer membrane protein alone. BALB/c mice were immunized with purified fusion protein P1-HspBU Com1-HspBC-HspB-SP34. The mice were immunized with the strain of Coxella bassii. Finally, the load of Cocks' body in the spleen of the attacked mice was analyzed by quantitative PCR. The results of many experiments showed that the amount of splenic inoculation of mice immunized with the fusion protein was significantly lower than that of mice immunized with outer membrane protein alone, indicating that the heat shock fusion protein P1-HspBmember Com1-HspB was significantly lower than that of mice immunized with outer membrane protein alone. The immune protective effect of HspB-SP34 was better than that of P _ (1) C _ (1) P _ (34) HspB alone. Conclusion: the immunogenicity and immunological protection of P1-HspBU Com1-HspB-SP34 fusion protein produced by fusion of membrane surface protein of Cox's body and heat shock protein B are significantly stronger than that of single outer membrane protein. The results indicated that heat shock protein B (HSPB) had a significant immune enhancement effect, and the fusion protein P1-HspBU Com1-HspBC1-HspB-SP34 could be used as a candidate for the development of Q fever molecular vaccine.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378
【参考文献】
相关期刊论文 前1条
1 魏文进,温博海,邱玲,牛东升,陈梅玲,高宁;贝氏柯克斯体34×10~3重组外膜蛋白的免疫保护性[J];军事医学科学院院刊;2004年04期
本文编号：2328222
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