牛型布鲁氏菌苹果酸脱氢酶酶学性质及其免疫学特性研究

发布时间：2018-03-21 07:00

本文选题：牛型布鲁氏菌　切入点：苹果酸脱氢酶　出处：《西南大学》2012年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella)引起的一种人兽共患慢性细菌性传染病,该病在世界范围内广泛流行并严重危害着人畜健康。布鲁氏菌在宿主体内生存能力极强,在巨噬细胞内具有极强的繁殖能力以及防止杀伤的自我保护能力,但其不具有经典的毒力因子且不能引起强烈的先天性免疫,因而给布鲁氏菌的防治带来了很大的困难。体内诱导基因涉及病原菌在体内的生存和致病过程,本研究对本实验室筛选、鉴定的布鲁氏菌体内诱导抗原—苹果酸脱氢酶的酶学特性及生物学功能开展研究,探讨MDH在布鲁氏菌致病过程的机制。本研究以牛型布鲁氏菌A19株基因组为模板,通过PCR技术扩增出牛型布鲁氏菌MDH的编码基因Malate dehydrogenase (mdh),测序结果表明,该基因大小为936bp,与NCBI上公布的布鲁氏菌2308(登录号：NC_007618.1)序列100%同源。PCR产物经酶切后与表达载体pET-28a(+)连接,成功构建pET-28a-MDH重组质粒,转化入大肠杆菌BL-21(DE3),经IPTG诱导表达后,成功表达大小约为36KDa的融合蛋白His-MDH。通过优化表达条件,His-MDH主要以可溶性方式表达,利用表达载体pET-28a(+)上的6His-Tag标记,通过Ni+亲和层析柱成功对His-MDH进行纯化,为进一步研究该蛋白的酶学性质及免疫学特性奠定了基础。苹果酸脱氢酶(MDH)是三梭酸循环中的一个关键酶,MDH以NAD+为辅因子,催化苹果酸盐与草酰乙酸盐的相互转化。本研究对表达、纯化的布鲁氏菌MDH的酶学性质进行了初步研究,结果表明：在体外催化苹果酸盐与草酰乙酸盐的酶促反应中,最适pH值为6.0,反应最适温度为42℃,在50℃以下酶的稳定性较好,Zn2+、Pb2+和Cu2+对酶有明显的抑制作用。酶动力学参数测得Km为0.67(mM),Vmax为0.91 (umol ml-1 min-1)。生物信息学分析表明,该酶为四聚体,单体存在N一端的NAD结合区、催化区及C—端尾区三个结构域,同时也存在与底物结合位点,疏水结构易形成酶的口袋结构,通过对不同细菌MDH的酶学活性位点分析,对布鲁氏菌MDH可能存在的酶活位点进行预测。以重组质粒pET-28a-MDH为模板,采用重叠引物延伸PCR技术在布鲁氏菌mdh基因中分别引入六个点突变(R89L、D149V、R152L、H176P、D178V、A231V)后与表达载体pET-28a(+)连接,转入大肠杆菌BL21(DE3)中,通过诱导表达与纯化,成功获得六个点突变的重组MDH。通过对MDH酶学活性的比较测定,结果发现除D178V点突变对酶活基本无影响外,其它五个点突变都可以使酶活完全丧失。 Western Blot检测结果表明,重组His-MDH能与布鲁氏菌阳性牛血清发生特异性反应,表明MDH具有免疫原性。用His-MDH作为包被抗原,对临床收集的30份布鲁氏菌阳性牛血清进行ELISA检测,结果所有被检血清均为阳性反应,表明MDH可作为潜在的用于牛型布鲁氏菌感染诊断的靶蛋白。生物信息学对MDH的氨基酸一级序列抗原性、亲水性、表面可及性以及柔韧性等指标的分析,一共预测了8条B细胞线性表位。此外,对MDH的亚定位结果表明,MDH为膜相关蛋白；纤连蛋白和纤连蛋白溶酶原结合活性分析以及布鲁氏菌感染的Hela细胞实验表明：MDH蛋白具有纤连蛋白和纤连蛋白溶酶原结合活性,参与布鲁氏菌对宿主细胞的入侵过程。本研究通过对布鲁氏菌苹果酸脱氢酶酶学性质以及免疫学特性研究,对阐述MDH参与布鲁氏菌致病过程中的作用提供依据,为进一步基于MDH在牛型布鲁氏菌在诊断、药物及疫苗研究提供理论基础。
[Abstract]:Brucellosis (Brucellosis) by Brucella (Brucella) caused by a chronic zoonotic bacterial infectious disease, the disease in the world widely popular and serious harm to human and animal health. In vivo Brucella survival ability is very strong, has the very strong ability of reproduction and to prevent the destruction of self-protection capability in macrophages inside, but it does not have the classic virulence factors and can not be caused by innate immunity strong, so to the prevention of brucellosis has brought great difficulties. In vivo induced genes involved in pathogen survival and pathogenesis in vivo, the research on the screening laboratory, to carry out research on the identification of Brucella in vivo induced antigen - malate dehydrogenase enzymatic properties and biological function of MDH in the mechanism of Brucella virulence process.
In this study, bovine Brucella strain A19 genome as template, amplified by PCR from bovine Brucella MDH gene encoding Malate dehydrogenase (MDH), gene sequencing results showed that the size of 936bp, and NCBI released 2308 Brucella (accession number: NC_007618.1) 100% sequence homology.PCR products digested with expression the vector pET-28a (+) is successfully constructed pET-28a-MDH plasmid was transformed into Escherichia coli BL-21 (DE3), induced by IPTG, expression of 36KDa fusion protein was about His-MDH. by optimizing the expression conditions, His-MDH is mainly expressed in the soluble form, using the expression vector pET-28a (+) 6His-Tag labeled by Ni+. Affinity chromatography was used to purify His-MDH successfully, which laid the foundation for the further study of enzymatic properties and immunological properties of the protein.
Malate dehydrogenase (MDH) is a key enzyme of three carboxylic acid cycle, MDH to NAD+ cofactor, catalyzes the interconversion of malate and oxaloacetate. The study on the expression and enzymatic properties of the purified MDH of Brucella was studied. The results showed that in vitro catalytic malate and oxalacetate the enzymatic reaction, the optimum pH value was 6, the optimum temperature is 42 DEG C below 50 DEG C in reaction, enzyme stability is better, Zn2+, Pb2+ and Cu2+ have obvious inhibitory effect on the enzyme. The enzyme kinetic parameters measured Km was 0.67 (mM), Vmax 0.91 (umol ml-1 min-1).
Bioinformatics analysis showed that the enzyme is a tetramer with four monomers of N end of the NAD binding region, catalytic domain and C - end of three domains of tail area, there is also a binding site and substrate structure to form hydrophobic pocket structure of enzyme, the enzyme activity of different bacterial MDH site analysis that may exist on Brucella MDH activity sites were predicted. The recombinant plasmid pET-28a-MDH as template, using overlapping primer extension PCR in Brucella MDH gene were introduced into six point mutations (R89L, D149V, R152L, H176P, D178V, A231V) and the expression vector pET-28a (+) connected into Escherichia coli BL21 (DE3), and purified by inducing the expression of recombinant MDH., successfully obtained six point mutation by comparison on Determination of MDH enzyme activity. The results show that in addition to D178V point mutation has no effect on enzyme activity, the other five point mutations can make the enzyme completely lost Lost.
Western Blot assay showed that the recombinant His-MDH could react specifically with Brucella positive bovine serum, showed that MDH had immunogenicity. Using His-MDH as antigen ELISA was detected in 30 Brucella positive bovine serum collection of clinical results, all tested serum were positive, indicating that MDH can be used as a target for protein in the diagnosis of bovine type Brucella infection potential. The bioinformatics of MDH amino acid sequences analysis of hydrophilicity, antigenicity, surface accessibility and flexibility index, a total of 8 predicted B cell linear epitopes. In addition, the sub localization of MDH showed that MDH is a membrane associated protein fiber; fibronectin and fibronectin plasminogen binding activity analysis and Brucella infection Hela cell experiments showed that MDH protein with fibronectin and fibronectin plasminogen binding activity in Bruce strains of The invasion process of the host cell.
This study provides a basis for elucidating the role of MDH in the pathogenesis of Brucella by studying the enzymatic properties and immunological characteristics of Brucella malate dehydrogenase, and provides a theoretical basis for further research based on MDH in the diagnosis, drug and vaccine of Brucella bovis.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378;S852.61

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