【作者】 龚薇；

【导师】 何凤田；

【作者基本信息】 第三军医大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 第一部分HMGB1酸性尾端抗菌相关功能位点解析研究目的:高迁移率族蛋白B1(high-mobility group box-1,HMGB1)属高迁移率族蛋白超家族成员,由A box、B box及酸性尾端三个结构域组成,其广泛存在于真核细胞核内,也可存在于胞浆及细胞外等多个部位,在机体正常生理活动维持和某些疾病状态下发挥多种重要功能。相对于其他功能而言,抗菌活性是HMGB1的一项新功能。我们前期在对HMGB1功能机制进行研究时发现:重组人HMGB1具有抗菌活性,然而缺失由30个氨基酸残基组成的酸性尾端的突变体蛋白则丧失抗菌能力;另外,我们还证实了单独的A box和B box不具有抗菌活性。因此,HMGB1酸性尾端对其抗菌活性的发挥至关重要。但是,针对我们获得的这一新发现,至少还有两个问题需进一步探讨:酸性尾端区域中参与抗菌作用的详细功能位点尚待解析;单独的酸性尾端是否即有抗菌能力还有待鉴定。本部分实验旨在寻找HMGB1酸性尾端中抗菌相关功能位点,并观察单独酸性尾端的抗菌活性。方法及结果:1.人HMGB1酸性尾端不同位点缺失突变体的制备(1)用已克隆于pUC19载体中测序正确的、编码人HMGB1的cDNA为模板,经常规PCR或一步反向PCR致突变策略分别扩增得到11种人HMGB1酸性尾端不同位点缺失突变体蛋白的编码序列,并成功的构建了重组质粒:pUC19/mHMGB1 -211-215、pUC19/mHMGB1 -206-215、pUC19/mHMGB1 -201-215、pUC19/mHMGB1 -196-215、pUC19/mHMGB1 -191-215、pUC19/mHMGB1 -186-200、pUC19/mHMGB1 -196-210、pUC19/mHMGB1 -196-205、pUC19/mHMGB1 -198-207、pUC19/mHMGB1 -201-210、pUC19/mHMGB1 -201-205。(2)将上述测序正确的各突变体编码序列分别亚克隆到适于表达毒性蛋白的原核表达载体pQE-80L中,成功地构建了重组原核表达质粒pQE-80L/mHMGB1 -211-215、pQE-80L/mHMGB1 -206-215、pQE-80L/mHMGB1 -201-215、pQE-80L/mHMGB1 -196-215、pQE-80L/mHMGB1 -191-215、pQE-80L/mHMGB1 -186-200、pQE-80L/mHMGB1-196-210、pQE-80L/mHMGB1 -196-205、pQE-80L/mHMGB1 -198-207、pQE-80L/mHMGB1 -201-210、pQE-80L/mHMGB1 -201-205。(3)将上述重组原核表达质粒分别转化于大肠杆菌DH5α,在终浓度0.5mmol/L的IPTG、37℃诱导表达3h。SDS-PAGE分析可见,目的蛋白相对分子质量均在30kDa- 25kDa之间,均主要以分泌形式表达。经Ni2+-NTA系统有效纯化后,我们成功的制备了11种人HMGB1酸性尾端不同位点缺失的突变体蛋白:mHMGB1 -211-215、mHMGB1 -206-215、mHMGB1 -201-215、mHMGB1 -196-215、mHMGB1 -191-215、mHMGB1 -186-200、mHMGB1 -196-210、mHMGB1 -196-205、mHMGB1 -198-207、mHMGB1 -201-210、mHMGB1 -201-205。2.人HMGB1酸性尾端不同位点缺失突变体抗菌功能的检测与比较体外抗菌实验(试管稀释法、琼脂扩散法)结果证实:11种人HMGB1酸性尾端不同位点缺失突变体中mHMGB1 -211-215、mHMGB1 -206-215、mHMGB1 -186-200对金黄色葡萄球菌、大肠杆菌JM109、ATCC25922、DH5α具有不同程度的抗菌活性,其中对DH5α抗菌活性最弱;而对绿脓杆菌则无此抗菌活性。其余突变体抗菌活性均消失。上述实验结果表明:人HMGB1酸性尾端中201-205氨基酸残基对其抗菌功能的发挥至关重要。3.人工合成的人HMGB1酸性尾端抗菌活性研究(1)人工合成30个氨基酸残基组成的人HMGB1酸性尾端肽段。(2)体外抗菌实验(试管稀释法、琼脂扩散法)结果显示:该肽段对金黄色葡萄球菌、大肠杆菌JM109、ATCC25922、DH5α具有不同程度的抗菌活性,其中对DH5α抗菌活性最弱;而对绿脓杆菌则无此抗菌活性。因此,单独的人HMGB1酸性尾端肽段即具有抗菌活性。结论:1.人HMGB1酸性尾端中201-205氨基酸残基在抗菌活性的发挥中起至关重要的作用。2.人工合成30个氨基酸残基组成的人HMGB1酸性尾端肽段对部分细菌具有明确的抗菌活性,从另一侧面证实HMGB1的酸性尾端对其抗菌功能的发挥至关重要。第二部分HMGB1为靶点的抗炎措施研究研究目的:HMGB1是一种具有宽治疗窗口期的关键炎症因子,在多种急、慢性炎症疾病的发生和发展过程中起着极为重要的作用,目前以该分子为靶标的抗炎措施越来越受到人们的关注。现有研究证实,其发挥致炎作用的主要功能域为B box。然而,体外表达的单独A box能抑制HMGB1致炎活性,有明确的抗炎功能。同时,酸性尾端具有调节A box、B box结构和功能的作用。如果能将HMGB1具有抗炎功能的A box与具有抗菌功能的酸性尾端融合就有可能得到兼具抗炎、抗菌双功能的衍生分子。又有研究显示,被分泌至细胞外的HMGB1除是一种重要的炎症细胞因子外,还参与激活机体自身免疫系统,与多种自身免疫性疾病密切相关。中药提纯单体青藤碱(sinomenine,SIN)具有抗炎、免疫抑制等药理作用,广泛应用于临床治疗类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、干燥综合征(SS)等慢性炎性自身免疫性疾病,并取得较好疗效。然而,SIN是否可影响HMGB1表达目前尚未见报道。本部分实验以HMGB1为靶点,制备由HMGB1 A box及酸性尾端组成的兼具抗炎、抗菌双功能的衍生分子;同时研究SIN对HMGB1表达的影响,并初步探讨其可能机制。方法及结果:1.HMGB1 A box和酸性尾端融合分子的制备(1)以克隆于pUC19载体中测序正确的、编码人HMGB1的cDNA为模板,经一步反向PCR,在缺失B box编码序列的同时,将A box与酸性尾端的编码序列直接相连(此连接为刚性连接),命名为pUC19/rHMGB1 A+T。(2)再用上述克隆于pUC19载体上测序正确的、处于刚性连接的A box与酸性尾端编码序列为模板,经多次一步反向PCR在A box与酸性尾端编码序列之间引入一个柔性连接子(Gly4Ser)3的编码序列(此连接为柔性连接),命名为pUC19/rHMGB1 A+Linker+T。(3)将测序正确的A box与酸性尾端刚性、柔性连接融合分子编码序列分别亚克隆于原核表达载体pQE-80L,分别命名为pQE-80L/rHMGB1 A+T、pQE-80L/rHMGB1 A+Linker+T,并在大肠杆菌DH5α中进行诱导表达上述融合蛋白。SDS-PAGE分析可见,两融合蛋白的相对分子质量分别约为16kDa和18kDa。经Ni2+-NTA纯化系统,两融合蛋白得到有效纯化,纯化后的目的蛋白在SDS-PAGE上均呈现单一条带。2.HMGB1 A box和酸性尾端融合分子抗炎功能的鉴定与比较(1)体外抗炎实验证实,两融合蛋白与单独A box类似均能抑制重组HMGB1诱导的人单核细胞THP-1分泌炎症因子肿瘤坏死因子a(TNF-a)和白细胞介素6(IL-6),两融合蛋白抑制炎症因子作用均比单独的A box强,且柔性连接的融合蛋白抗炎能力是三者中最强的。(2)在LPS诱导的小鼠内毒素血症模型(模拟内毒素引起的系统性炎症)中两融合蛋白均能降低血清炎症因子TNF-a、IL-6的浓度并能提高小鼠的存活率;同体外实验结果类似柔性连接的融合蛋白降低血清中炎症因子浓度及提高小鼠存活率作用更强。3.HMGB1 A box和酸性尾端融合分子抗菌功能的检测与比较体外抗菌实验(试管稀释法、琼脂扩散法)结果显示,A box与酸性尾端刚性连接和柔性连接两融合蛋白均能不同程度抑制金黄色葡萄球菌、大肠杆菌(JM109、ATCC25922、DH5α)生长,但二者的抑制细菌生长能力并无统计学差异;均对绿脓杆菌无抗菌活性。4.盐酸青藤碱抑制基础水平和LPS诱导的HMGB1表达(1)在人内皮细胞EA.hy926中盐酸青藤碱可抑制基础水平的HMGB1 mRNA和蛋白质表达,且呈剂量依赖性。(2)在人内皮细胞EA.hy926中盐酸青藤碱可抑制LPS诱导水平的HMGB1 mRNA和蛋白质表达,且呈剂量依赖性。5.盐酸青藤碱抑制HMGB1基因启动子活性(1)成功克隆到具有活性的HMGB1基因5’上游启动子区域(-2484~+200),并构建长度不同的HMGB1基因启动子区域截短突变体(-1998~+200、-1598~+200、-1198~+200、-798~+200、-397~+200)报告基因检测质粒。(2)荧光素酶(Luc)报告基因检测实验结果知,盐酸青藤碱可抑制-2484~+200、-1998~+200、-1598~+200、-1198~+200报告基因质粒启动子活性,但对-798~+200、-397~+200报告基因质粒启动子活性无影响,此结果表明HMGB1基因5’上游启动子区域-1198~-798序列在青藤碱抑制HMGB1转录中起至关重要的作用。结论:1.人HMGB1 A box与酸性尾端刚性连接和柔性连接两种衍生分子均是兼具抗炎、抗菌的双功能分子;酸性尾端可增强A box的抗炎活性,且柔性连接的融合分子抗炎活性强于刚性连接的融合分子,但其二者在抗菌活性方面无差异;2.青藤碱可抑制基础水平及LPS诱导的HMGB1表达,且HMGB1基因5’上游启动子区域-1198~-798序列与青藤碱抑制HMGB1转录密切相关。更多还原

【Abstract】 Part I Analysis of antibacterial-associated functional sites in human HMGB1 acidic tailObjective: High-mobility group box 1(HMGB1) belongs to high-mobility group superfamily named for its characteristic rapid mobility in polyacrylamide gel electrophoresis (PAGE). Structurally, HMGB1 has 215 amino acid residues, including three main functional domains: A box, B box and C-terminal acidic tail (C tail). HMGB1 has been shown multiple important functions in nucleus, cytoplasm even extracellular enviroment. In physiological situation, as a potent antibacterial protein, HMGB1 is an important part of innate immunity defensive barrier of the human body that can resist bacterial infection in vivo. But the functional domain for this new effect is presently unknown. In our previous reseaches, we identified that the full-length recombination human HMGB1 presented antibacterial activity, nevertheless, the A box and the B box domains of the molecule and the truncated HMGB1 lacking its C tail failed to inhibit bacterial multiplication, which demonstrated that the C tail was the key domain for the antibacterial activity of HMGB1. But there are still remained some futhur reseaches such as which region in C tail is mainly responsible for the antibacterial activity of HMGB1, and whether the acidic tail alone peptide has antibacterial function.Methods and Results: The encoding fragements of eleven different deleted mutants in C tail of human HMGB1 were constructed by PCR or one-step opposite-direction PCR, which were laking its amino acid residues 211-215, 206-215, 201-215, 196-215, 191-215, 186-200, 196-210, 196-205, 198-207, 201-210, 201-205 respectively. They were consistent with the sequence reported in GenBank. These mutants were successfully expressed in the prokaryotic expression systerm. The relative molecular masses of them were about from 30kDa to 25kDa. The expressed proteins were purified by Ni2+-NTA chromatography. SDS-PAGE analysis showed that the target proteins were highly purified. The acidic tail peptide alone (C peptide) was synthesized. The antibacterial activities of these mutants and C peptide to Staphylococcus aureus (SA), E.coli (JM109, ATCC25922, DH5α), P. aeruginosa (PA) were compared by bacteria dilution in the test tubes and dispersion method. The results showed that the truncated mutants which lacking its amino acid residues 211-215, 206-215, 186-200 respectively and C peptide could inhibit the proliferation of SA, E.coli.JM109, ATCC25922 and DH5αefficiently in vitro. However, other mutants had no antibacterial function. Moreover, all of the mutants and C peptide couldn’t inhibit PA growth.Conclusion: We concluded that the amino acid residues 201-205 in C-terminal acidic tail region are the core functional site for the antibacterial activity of the molecule. And C peptide could exert antibacterial function. Part II Analysis of anti-inflammation strategies against HMGB1Objective: HMGB1 has been identified as a mediator of endotoxin lethality and become a new therapeutic target in recent years, which plays a critical role in the processes of many kinds of acute and chronic inflammation, including autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sj?gren Syndrome (SS) and so on. B box of HMGB1 is the inflammation functional domain. Interestingly, HMGB1 A box can antagonize HMGB1-induced inflammation. Moreover, the C tail contributed to the spatial structure of both A box and B box and regulated HMGB1 DNA binding specificity. However, it is unknown whether the C tail can enhance the anti-inflammatory activity of the A box, and whether the fusion proteins based on the A box and C tail have antibacterial and anti-inflammation activity. Then in order to investigate whether the novel fusion molecules have antibacterial activity and anti-inflammation activity, which consist of the human HMGB1 antibacterial acidic tail and A box possessing anti-inflammation action, we prepared two fusion molecules consisting of the human HMGB1 A box and acidic tail linked directly and by a flexible linker sequence (Gly4Ser)3 respectively. The effects of these fusion molecules were compared by antibacterial and anti-inflammation assays in vitro and in vivo.Sinominine, a purified alkaloid extracted from sinomenium acutum, has a variety of pharmacological effects including anti-inflammation, immunosuppression, and anti-angiogenesis, which is treated to RA, SLE. It is not sure whether sinominine can influence the expression of HMGB1. Finally, we tested whether sinomenine inhibits the expression of HMGB1.Methods and Results: The fusion molecules consisting of the human HMGB1 antibacterial acidic tail and A box possessing anti-inflammation action linked directly and by a flexible linker sequence (Gly4Ser)3 were successfully prepared. The relative molecular masses of the two fusion molecules were about 16kDa and 18kDa. Antibacterial assays showed that these two molecules had equal abilities to inhibit the proliferation of SA, E.coli.JM109, ATCC25922 and DH5αin vitro. They could inhibit TNF-a and IL-6 release in vitro and in vivo and increase the survival rate of endotoxemia mouse. And the flexible linker fusion molecule had more powerful anti-inflammation activity. We also detected that sinomenine could inhibit both basal and LPS stimulated HMGB1 expression in human endothelial cell EA.Hy926 by Real-time PCR and Western blotting. Morover, we cloned the promoter of HMGB1. Luciferase assay showed that sinomenine could suppress the transcription activity of HMGB1 promoter and tightly related with -1198～-798 of the promoter region.Conclusion: In summary, the novel fusion molecules have antibacterial and anti-inflammation activity. The fused C tail can enhance the anti-inflammatory effect of the A box. And sinomenine could inhibit HMGB1 expression by suppressing the transcription activity of -1198～-798 in HMGB1 promoter region.更多还原