貝氏柯克斯體外膜蛋白與熱休克蛋白融合蛋白的研制及其免疫原性的研究
發(fā)布時(shí)間：2018-11-12 21:08
【摘要】： 貝氏柯克斯體是一種專性細(xì)胞內(nèi)寄生菌，為重要人畜共患病Q熱的病原體，也是一種重要的生物戰(zhàn)劑和生物恐怖劑。Q熱疫苗接種是預(yù)防貝氏柯克斯體感染的最有效的措施。采用基因工程技術(shù)制備重組保護(hù)性抗原是研制Q熱分子疫苗的重要手段。 貝氏柯克斯體的外膜蛋白P1(分子量為29kDa)、Com1(分子量為27kDa)以及34kDa蛋白(SP34)是重要的保護(hù)性抗原，熱休克蛋白B(HspB)也是其主要表面抗原。另外許多研究證明熱休克蛋白具有的佐劑樣功能，能增強(qiáng)其它抗原的免疫保護(hù)效能。因此，本研究研制了貝氏柯克斯體HspB融合于P1、Com1、SP34外膜蛋白，制備了熱休克蛋白融合蛋白，并對(duì)這些融合蛋白的免疫原性和免疫保護(hù)性進(jìn)行評(píng)價(jià)。 采用分子克隆技術(shù)，從貝氏柯克斯體基因組中克隆P1、Com1、SP34外膜蛋白基因以及HspB基因；將外膜蛋白基因分別與HspB基因融合構(gòu)建了pQE30／p1-htpB、pQE30／com1-htpB、pET28a／htpB-sp34原核表達(dá)質(zhì)粒，原核表達(dá)質(zhì)粒轉(zhuǎn)化的大腸桿菌分別產(chǎn)生P1-HspB、Com1-HspB、HspB-SP34融合蛋白。免疫印跡分析顯示這些融合蛋白均可以與貝氏柯克斯體感染血清發(fā)生特異性的反應(yīng)，證明其具有良好的抗原反應(yīng)性。 采用親和層析的方法純化P1-HspB、Com1-HspB、HspB-SP34融合蛋白，用它們分別免疫BALB／c小鼠，分析其免疫原性。P1-HspB融合蛋白免疫小鼠不僅產(chǎn)生特異性IgG抗體的水平顯著高于單P1蛋白免疫小鼠，而且γ干擾素和IL-2細(xì)胞因子水平也顯著高于P1單個(gè)蛋白免疫。檢測Com1-HspB和HspB-SP34融合蛋白免疫小鼠后的抗體水平，結(jié)果同樣表明融合蛋白誘導(dǎo)機(jī)體產(chǎn)生特異性抗體的能力顯著好于外膜蛋白單獨(dú)免疫。 將純化的P1-HspB、Com1-HspB、HspB-SP34融合蛋白分別免疫BALB／c小鼠，用貝氏柯克斯體毒株對(duì)免疫小鼠進(jìn)行攻擊，最后用定量PCR分析受攻擊小鼠脾臟的貝氏柯克斯體載量。多次試驗(yàn)的結(jié)果顯示融合蛋白免疫小鼠的貝氏柯克斯體攻擊后脾臟荷菌量顯著低于單獨(dú)外膜蛋白免疫小鼠，說明熱休克融合蛋白P1-HspB、Com1-HspB、HspB-SP34的免疫保護(hù)效果明顯好于P1、Com1、SP34、HspB單獨(dú)免疫。 結(jié)論：貝氏柯克斯體膜表面蛋白與熱休克蛋白B融合后產(chǎn)生的P1-HspB、Com1-HspB、HspB-SP34融合蛋白的免疫原性和免疫保護(hù)性顯著強(qiáng)于單個(gè)外膜蛋白，，說明熱休克蛋白B具有顯著的免疫增強(qiáng)作用。P1-HspB、Com1-HspB、HspB-SP34融合蛋白可作為研制Q熱分子疫苗的候選分子。
[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 緯 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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378
本文編號(hào)：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 緯 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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 魏文進(jìn),溫博海,邱玲,牛東升,陳梅玲,高寧;貝氏柯克斯體34×10~3重組外膜蛋白的免疫保護(hù)性[J];軍事醫(yī)學(xué)科學(xué)院院刊;2004年04期
本文編號(hào)：2328222
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