副结核分枝杆菌MAP1068胞外蛋白酶的特性研究
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摘要
为研究副结核分枝杆菌(MAP) 1068蛋白的特性,本研究以MAP参考株K10基因组为模板,PCR扩增其map1068基因,构建p ET28a-map1068重组载体,将其转化BL21后诱导表达并纯化MAP1068蛋白。利用纯化的重组蛋白免疫(100μg/只/0.1 m L)小鼠制备多克隆抗体,应用该抗体对MAP1068蛋白进行亚细胞定位。以酪蛋白为底物测定MAP1068蛋白的酶活性。结果显示,MAP1068蛋白以包涵体形式表达;经变性、复性、亲和层析纯化后获得目的蛋白;将该蛋白免疫小鼠后其血清抗体效价可达1∶204 800;该蛋白主要定位于MAP细胞壁,可降解酪蛋白,为胞外蛋白酶。本实验为进一步研究MAP的致病机制奠定了基础。
In order to explore the characteristics of MAP1068 protein from Mycobacterium avium subsp. Paratuberculosis(MAP), this experiment used the MAP reference strain K10 genome as a template to specifically amplify the map1068 gene, which was subcloned into to the pET28 a vector after digestion. The resulting recombinant vector pET28 a-map1068 was then transformed to E.coli BL21 to induce the expression of MAP1068 protein. After purification the recombinant protein was used to immunize(100μg/0.1 mL) mice to prepare polyclonal antibody against MAP1068 protein, and applied the antibody for subcellular localization. The enzymatic activity of the MAP1068 protein was assayed using casein as the substrate. The results showed that MAP1068 protein was expressed in the form of inclusion bodies, and target protein was obtained by denaturing renaturation and affinity chromatography. After immunized mice, 1∶204,800 high titer antibody was obtained. The protein is mainly localized on the cell wall and it is an extracellular protein possessing the enzymatic activity of casein degradation. The results of this experiment lay the foundation for further study of the pathogenic mechanism of MAP.
In order to explore the characteristics of MAP1068 protein from Mycobacterium avium subsp. Paratuberculosis(MAP), this experiment used the MAP reference strain K10 genome as a template to specifically amplify the map1068 gene, which was subcloned into to the pET28 a vector after digestion. The resulting recombinant vector pET28 a-map1068 was then transformed to E.coli BL21 to induce the expression of MAP1068 protein. After purification the recombinant protein was used to immunize(100μg/0.1 mL) mice to prepare polyclonal antibody against MAP1068 protein, and applied the antibody for subcellular localization. The enzymatic activity of the MAP1068 protein was assayed using casein as the substrate. The results showed that MAP1068 protein was expressed in the form of inclusion bodies, and target protein was obtained by denaturing renaturation and affinity chromatography. After immunized mice, 1∶204,800 high titer antibody was obtained. The protein is mainly localized on the cell wall and it is an extracellular protein possessing the enzymatic activity of casein degradation. The results of this experiment lay the foundation for further study of the pathogenic mechanism of MAP.
引文
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[2] Squeglia F, Ruggiero A, Romano M, et al. Mutational and structural study of Rip A, a key enzyme in Mycobacterium tuberculosis cell division:evidence for the L-to-D inversion of configuration of the catalytic cysteine[J]. Acta Crystallogr D Biol Crystallogr, 2014, 70(9):2295-2300.
[3] Spoerry C, Seele J, Valentin-weigand P, Baums C G, et al.Identification and characterization of IgdE, a novel IgG-degrading protease of Streptococcus suis with unique specificity for porcine IgG[J]. JBC, 1975, 291(15):7915-7925.
[4]宋军科,才学鹏,骆学农,等.寄生虫半胱氨酸蛋白酶研究进展[J].动物医学进展,2011,32(12):95-98.
[5]潘耀谦,王帅,李瑞珍,等.兔豆状囊尾拗囊液蛋白的纯化与抗原性鉴定[J].中国兽医学报,2018,(1):88-95.
[6] Rudner D Z, Losick R. Protein subcellular localization in bacteria[J]. Cold Spring Harb Perspect Biol, 2010, 2(4):a000307.
[7]曾胤新,蔡明宏,陈波,等.一株产蛋白酶南极耐冷细菌的筛选及研究[J].生物技术,2001,11(2):17-19.
[8] Dutt K, Meghwanshi G K, Gupta P, et al. Role of casein on induction and enhancement of production of a bacterial milk clotting protease from an indigenously isolated Bacillus subtilis[J].Lett Appl Microbiol, 2010, 46(5):513-518.
[9] Dang Guang-hui, Cao Jun, Cui Ying-ying, et al. Characterization of Rv0888, a novel extracellular nuclease from Mycobacterium tuberculosis[J]. Sci Rep, 2016, 6:19033.