HMGB1酸性尾端对其抗菌活性的影响

发布时间：2018-05-13 13:25

  本文选题：高迁移率族蛋白B1 + 抗菌活性　； 参考：《第三军医大学》2006年硕士论文

【摘要】： 高迁移率族蛋白B1(high-mobility group box-1,HMGB1)是广泛存在于真核生物细胞核内的染色体结合蛋白,其也存在于细胞质和细胞外,主要由A box、B box及酸性尾端三个结构域组成,具有多种生理和病理学作用。近年来研究表明,抗菌活性是HMGB1的一项新功能,然而其抗菌活性产生的机理尚未知。如能弄清HMGB1抗菌功能的结构基础,明确其参与抗菌作用的功能区域,不仅对深入探讨HMGB1抗菌活性发挥的具体功能位点及其结构与功能之间的关系具有重要的理论指导意义,而且可为进一步揭示HMGB1的抗菌机制奠定良好的基础。有文献报道天然人HMGB1有明确的抗菌活性,我们在实验过程中亦发现重组人HMGB1在适于表达“毒性”蛋白的原核表达系统pQE-80L/DH5α中表达量较低,而在其余一些原核表达系统中则不能表达;并且重组人HMGB1 A box、B box均无抗菌活性,我们推测HMGB1的酸性尾端对其抗菌活性的发挥可能有影响。为了证实这一推测,本实验表达、纯化了重组人HMGB1 A box及B box蛋白,继而克隆、表达并纯化了重组人HMGB1及其缺失酸性尾端的突变体蛋白;通过试管稀释法、琼脂扩散法两种抗菌实验观察并比较各组蛋白对金黄色葡萄球菌、大肠杆菌(JM109、ATCC25922、DH5α)、绿脓杆菌的抗菌活性。为了解与人HMGB1具有高同源性的小鼠HMGB1是否也具有相同的抗菌活性,进一步克隆、表达并纯化重组小鼠HMGB1及其缺失酸性尾端的突变体蛋白,运用同样的抗菌实验观察并比较该两蛋白的抗菌活性。 主要结果: 1.表达人HMGB1 A box与DHFR融合蛋白、人HMGB1 B box与DHFR融合蛋白及DHFR蛋白。经Ni2+-NTA纯化系统,各目的蛋白得到了有效纯化。SDS-PAGE分析可见,分别在相对分子质量约为36×103、35×103、26×103处呈现单一条带。 2.分别经RT-PCR成功地克隆了编码人、小鼠HMGB1的cDNA(即hHMGB1 cDNA、mHMGB1 cDNA)。测序后经查对,与GenBank上登录的编码相应人、小鼠HMGB1的cDNA序列完全一致。 3.分别以获得的pUC19/hHMGB1、pUC19/mHMGB1为模板,经PCR致突变法获得其各自缺失酸性尾端的突变体DNA,分别命名为pUC19/mhHMGB1、pUC19/
[Abstract]:High mobility group protein B1(high-mobility group box-1 (HMGB1) is a chromosomal binding protein widely found in the nucleus of eukaryotes. It also exists in cytoplasm and extracellular, and is composed of three domains, A box B box and acid tail. It has many physiological and pathological functions. Recent studies have shown that antimicrobial activity is a new function of HMGB1, but the mechanism of its antibacterial activity has not been known. If we can make clear the structural basis of the antibacterial function of HMGB1 and clarify the functional region of its antimicrobial activity, it is not only of great theoretical significance to study the specific functional sites and the relationship between the structure and function of the antibacterial activity of HMGB1. And it can lay a good foundation for further revealing the antibacterial mechanism of HMGB1. It has been reported that natural human HMGB1 has specific antibacterial activity. We also found that the expression of recombinant human HMGB1 in prokaryotic expression system pQE-80L/DH5 伪, which is suitable for the expression of "toxic" protein, was low. Moreover, the recombinant human HMGB1 A boxB box had no antimicrobial activity. We speculate that the acid end of HMGB1 may have an effect on its antimicrobial activity. In order to confirm this hypothesis, the recombinant human HMGB1 A box and B box proteins were purified, then cloned, and the mutant proteins of recombinant human HMGB1 and its missing acid tail terminal were expressed and purified. The antimicrobial activity of two groups of proteins against Staphylococcus aureus, Escherichia coli JM109, ATCC25922 DH5 伪 and Pseudomonas aeruginosa were observed and compared by Agar diffusion method. In order to find out whether murine HMGB1 with high homology with human HMGB1 has the same antibacterial activity, we further clone, express and purify the recombinant mouse HMGB1 and the mutant protein with missing acid tail end. The antimicrobial activity of the two proteins was observed and compared with the same antimicrobial experiment. Main results: 1. Human HMGB1 A box and DHFR fusion protein, human HMGB1 B box and DHFR fusion protein and DHFR protein were expressed. By Ni2-NTA purification system, all the target proteins were effectively purified. SDS-PAGE analysis showed that there was a single band at the relative molecular weight of 36 脳 10 ~ (3) ~ (35) 脳 10 ~ (3) C ~ (26 脳 10 ~ (3). 2. The cDNAs of human and mouse HMGB1 (hHMGB1 cDNA-mHMGB1) were cloned successfully by RT-PCR. After sequencing, the cDNA sequence of mouse HMGB1 was identical to that of the corresponding human registered on GenBank. 3. Using the obtained pUC19 / hHMGB1 / pUC19 / mHMGB1 as template, the mutagenesis method of PCR was used to obtain the mutant DNA of their respective missing acid tail, named pUC19 / mhHMGB1 / pUC19 / 1, respectively, and named as pUC19 / mHMGB1 / mHMGB1, respectively.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341
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本文编号：1883335