本文選題：高遷移率族蛋白B1 + 抗菌活性��； 參考：《第三軍醫(yī)大學(xué)》2006年碩士論文

【摘要】： 高遷移率族蛋白B1(high-mobility group box-1,HMGB1)是廣泛存在于真核生物細(xì)胞核內(nèi)的染色體結(jié)合蛋白,其也存在于細(xì)胞質(zhì)和細(xì)胞外,主要由A box、B box及酸性尾端三個(gè)結(jié)構(gòu)域組成,具有多種生理和病理學(xué)作用。近年來(lái)研究表明,抗菌活性是HMGB1的一項(xiàng)新功能,然而其抗菌活性產(chǎn)生的機(jī)理尚未知。如能弄清HMGB1抗菌功能的結(jié)構(gòu)基礎(chǔ),明確其參與抗菌作用的功能區(qū)域,不僅對(duì)深入探討HMGB1抗菌活性發(fā)揮的具體功能位點(diǎn)及其結(jié)構(gòu)與功能之間的關(guān)系具有重要的理論指導(dǎo)意義,而且可為進(jìn)一步揭示HMGB1的抗菌機(jī)制奠定良好的基礎(chǔ)。有文獻(xiàn)報(bào)道天然人HMGB1有明確的抗菌活性,我們?cè)趯?shí)驗(yàn)過(guò)程中亦發(fā)現(xiàn)重組人HMGB1在適于表達(dá)“毒性”蛋白的原核表達(dá)系統(tǒng)pQE-80L/DH5α中表達(dá)量較低,而在其余一些原核表達(dá)系統(tǒng)中則不能表達(dá);并且重組人HMGB1 A box、B box均無(wú)抗菌活性,我們推測(cè)HMGB1的酸性尾端對(duì)其抗菌活性的發(fā)揮可能有影響。為了證實(shí)這一推測(cè),本實(shí)驗(yàn)表達(dá)、純化了重組人HMGB1 A box及B box蛋白,繼而克隆、表達(dá)并純化了重組人HMGB1及其缺失酸性尾端的突變體蛋白;通過(guò)試管稀釋法、瓊脂擴(kuò)散法兩種抗菌實(shí)驗(yàn)觀察并比較各組蛋白對(duì)金黃色葡萄球菌、大腸桿菌(JM109、ATCC25922、DH5α)、綠膿桿菌的抗菌活性。為了解與人HMGB1具有高同源性的小鼠HMGB1是否也具有相同的抗菌活性,進(jìn)一步克隆、表達(dá)并純化重組小鼠HMGB1及其缺失酸性尾端的突變體蛋白,運(yùn)用同樣的抗菌實(shí)驗(yàn)觀察并比較該兩蛋白的抗菌活性。 主要結(jié)果: 1.表達(dá)人HMGB1 A box與DHFR融合蛋白、人HMGB1 B box與DHFR融合蛋白及DHFR蛋白。經(jīng)Ni2+-NTA純化系統(tǒng),各目的蛋白得到了有效純化。SDS-PAGE分析可見(jiàn),分別在相對(duì)分子質(zhì)量約為36×103、35×103、26×103處呈現(xiàn)單一條帶。 2.分別經(jīng)RT-PCR成功地克隆了編碼人、小鼠HMGB1的cDNA(即hHMGB1 cDNA、mHMGB1 cDNA)。測(cè)序后經(jīng)查對(duì),與GenBank上登錄的編碼相應(yīng)人、小鼠HMGB1的cDNA序列完全一致。 3.分別以獲得的pUC19/hHMGB1、pUC19/mHMGB1為模板,經(jīng)PCR致突變法獲得其各自缺失酸性尾端的突變體DNA,分別命名為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 偽, 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 偽 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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R341
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本文編號(hào)：1883335