【作者】 朱敏；

【导师】 赵跃然； 李建峰；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 一.立题目的和依据人高迁移率族B1 (high mobility group box 1, HMGB1)是含有215个氨基酸序列在哺乳动物细胞中广泛表达的高度保守的蛋白,因其相对分子质量小(M<30000),在聚丙烯酰胺凝胶电泳中快速迁移而被命名。HMGB1包括3个不同的功能区,其N-端包含2个结构域：HMBG A box和HMBG B box,均由约80个氨基酸残基构成,富含带正电荷的赖氨酸呈较强的碱性,为DNA非特异结合区；酸性C-末端包含超过30个连续的天冬氨酸和谷氨酸残基,籍以完成与其它蛋白质间相互作用及调节HMGB1与DNA结合。HMGB1蛋白在胞内外都有分布,根据其分布不同而发挥不同的生物学效应。人和小鼠的HMGB1只有两个氨基酸的差异,HMGB1缺陷的小鼠在出生后几个小时内就会死亡,说明HMGB1蛋白在维持细胞功能方面起着重要的作用。细胞内HMGB1参与构建核小体,该蛋白和DNA的小沟结合后改变DNA双螺旋构型,增加转录因子和DNA相互作用的亲和力调节DNA的转录,同时在DNA重组、复制和修复中亦起重要的辅助作用。细胞外HMGBI具有细胞因子特性,它与细胞分化、迁移、增殖、凋亡以及炎症反应的诱导等密切相关。尽管HMGB1蛋白缺少引导肽,且不能穿出内质网和高尔基细胞器,但仍可通过以下两种途径释放,一是由坏死但未凋亡的细胞释放,一是炎性因子刺激细胞后由细胞主动分泌,这些细胞如：巨噬细胞、NK细胞等。研究表明,HMGB1与炎细胞表面的RAGE、TLR-2、TLR-4等受体相互结合后可引发MAPK的磷酸化、核转录因子NF-κB和活化蛋白-1的核内转移,使许多炎性因子如IL-1β、TNF-α、IL-8等表达加强和释放增多而启动炎症反应或使炎症恶化。当细胞坏死或受损时,在TNF的作用下核内的HMGB1可释放到胞外,引发单核巨噬细胞分泌促炎因子；而促炎因子又可进一步促进HMGB1的分泌,形成正反馈环。在炎性反应的后期,这种正反馈效应对SLE等疾病炎性反应的维持具有重要的作用。在培养的巨噬细胞中分别加入HMGB1和B box蛋白,结果显示经二者刺激后均有较高水平的TNF-a的产生,使用抗B box蛋白的抗体后显示二者的TNF-a的产生水平明显降低,表明HMGB 1的致炎作用主要是由其B box结构域来行使,并且单独B box即可发挥致炎作用。同样在巨噬细胞中加入A box蛋白后可以明显降低HMGB1刺激后TNF-a的产生水平。表明A box蛋白起到了抑制炎症的作用成为HMGB1有效的拮抗剂。为探讨HMGB1 A box在SLE等自身免疫病中的治疗作用,我们进行了其表达载体构建、重组蛋白纯化和生物学活性的研究。二.方法和结果1.成熟人HMGB1 A box的基因克隆及序列分析根据GenBank报导的成熟人HMGB1编码序列对其进行优化,调整密码子适应指数(Codon Adaptation Index, CAI)使得CAI数值为0.95,增加编码序列中GC含量延长mRNA的半衰期,去除mRNA的二级结构,全基因合成质粒DNA pUC 57-HMGB1。根据优化后的HMGB1基因序列我们设计合成了扩增HMGB1 A box基因片段引物,分别在上下游引物5′端加上限制性酶切位点Nde I和Xho I,以优化后的HMGBl基因序列为模板,用Taq DNA聚合酶扩增HMGB1 A box基因片段。琼脂糖凝胶电泳分离PCR产物,胶回收试剂盒回收分离目的基因片段。将目的基因片段插入克隆载体pMD19-T,转化大肠杆菌DH5a,接种于含10Oug/mL氨苄青霉素的LB平板。菌落PCR和限制性酶谱分析筛选出阳性克隆,采用ABI3100-Avant Genetic Analyzer全自动DNA测序仪测序,结果显示,克隆的基因序列和优化后的成熟人HMGB1 A box基因序列完全一致。成功构建了人HMGB1 A box的重组质粒pMD19-T-HMGB1 A box.2.人HMGB1 A box的基因在大肠杆菌中的表达及重组蛋白的鉴定用Nde I和Xho I双酶切消化重组质粒pMD 19-T-HMGB 1 A box,低熔点琼脂糖凝胶电泳分离纯化HMGB1 A box基因片断,插入原核表达载体pQE-T7-2的相应位点构建重组pQE-A box表达载体。重组子转化大肠杆菌DH5α(EcoliDH5a),接种于含50ug/mL卡那霉素(Kanamycin)的LB平板,菌落PCR鉴定阳性重组。挑选阳性重组的单个菌落接种于含有50ug/mL卡那霉素的LB液体培养基中,提取质粒再转入大肠杆菌BL21感受态。挑选单个菌落接种于含有50ug/mL卡那霉素的LB液体培养基中,37℃震荡培养过夜,按1：100的比例稀释后接种于含卡那霉素抗性的LB液体培养基中震荡至对数生长期,加入IPTG诱导4小时,离心收集菌体行SDS-PAGE和Western blotting分析。SDS-PAGE结果显示诱导后pQE-A box阳性重组菌株在相对分子量(Mr)约为14000处可见一明显蛋白条带,与预期蛋白大小相符,图像扫描仪分析显示目的蛋白占菌体总蛋白的40%左右。Western blotting显示重组蛋白能与抗人HMGB1多克隆抗体和抗His-Tag多克隆抗体发生特异性反应,表明重组蛋白为特异性人HMGB1 A box蛋白。3.人HMGB1 A box蛋白的纯化及生物活性测定收集菌体,冰浴中超声裂解,离心收集上清。沉淀经0.5%triton X-100洗涤即为粗提的包涵体。7M尿素溶解包涵体离心后收集上清,超声后上清及包涵体裂解产物行SDS-PAGE。结果显示重组蛋白存在于上清中,提示HMGB1 A box以可溶性形式表达于菌株胞浆。经Ni2+-NTA层析柱纯化后SDS-PAGE检测纯化后的蛋白,结果显示重组蛋白HMGB1 A box纯度达90%以上。透析纯化后的蛋白去除咪唑等小分子物质。按文献制备细胞坏死液,免疫复合物(IC)由细胞坏死液与SLE患者血清以25：1的比例配制。U937细胞(3×108／L)接种于12孔板,1 ml／孔。实验分U937细胞+IC组,U937细胞+IC+HMGB1 A box组,每组设3个复孔。于37℃50ml/L CO2条件下孵育24 h。收集细胞,提取总RNA, RT-PCR检测细胞IFN-γ、BAFF及TNF-α水平,同时以GAPDH为内参照。PCR产物琼脂糖凝胶电泳图像经凝胶图像扫描仪扫描并分析,采用相对指数(Relative index,RI)计算BAFF、IFN-γ及TNF-a的表达水平。结果显示HMGB1 A box蛋白可有效抑制IC刺激单核细胞后BAFF、TNF-a及IFN-y的分泌,提示所制备的人HMGB1 A box蛋白具有显著的生物活性。三.结论1.成功克隆了人HMGB1 A box基因,序列分析显示,克隆的基因序列和优化后的基因序列一致。2.构建了人HMGB1 A box的原核表达载体,获得稳定表达的工程菌株。3.初步摸索优化了重组人pQE-A box的表达条件、包涵体提取步骤,蛋白初步纯化工艺。3.重组人HMGB1 A box蛋白的表达量占菌体总蛋白的40%左右,为特异性人HMGB1 A box蛋白。制备的人HMGB1 A box蛋白具有显著的生物活性。更多还原

【Abstract】 Background and objectiveHigh mobility group box1 (HMGB1) protein which contains 215 amino acid sequence of highly conserved can widely expressed in mammalian cells. Because of its relative molecular weight is small(M<30000),it can rapidly migrated in the poly-acrylamide gel electrophoresis. HMGB1 consists three different functional areas, its N-terminal has two non-specific DNA binding domains:HMGB Abox and HMGB B box. Each of them is constituted by about 80 amino acid residues, which are rich in positively charged lysine. Its acidic C-terminal contains more than 30 consecutive aspartate residues and glutamate residues in order to complete the interaction with other protein and regulate the combinant between HMBG1 and DNA. HMGB1 protein distributed both inside and outside of the cell, it can play different biological effects according to its distribution. There are only two amino acid of HMGB 1 difference between human and mouse, the mouse of HMGB 1 deficient will die within a few hours after birth that can told us HMGB 1 protein plays an important role in the maintenance of the cell function. Intracellular HMGB 1 participate in the construction of nuclear body, the protein combined to DNA minor groove to change configuration of the DNA double helix, increase the interaction of affinity between transcription factors and DNA, while in DNA recombinant replication and repair the protein also play an important role. Extracellular HMGB1 possess cytokine properties, it is closely associated with cellular differentiation, migration, proliferation, apoptosis and inflammation reaction. Although it lacks signal peptide and can not through out of the endocytoplasmic reticulum and Golgi cellular organelle, HMGB1 can still be taken into the extracellular through two routes, the one route is HMGB1 release from necrotic but not apoptotic cell, the other route is inflammatory factor stimulate cell,these cells such as:macrophage, cell natural killer cell and so on. Extracellular HMGB 1 must be combined with the corresponding membrane receptor in order to play its biological effects. Receptor for advanced glycation end produces (RAGE) has high-affinity to HMGB1 and is the main receptor of HMGB1. After combining with RAGE, HMGB1 can initiate phosphorylation of MAPK, the intranuclear shift of nucleus transcription factor NF-κB and the activated protein-1 in order to start inflammartory and deteriorates inflammartory by making many other inflammatory factors like IL-1β, TNF-a, IL-8 be expressed and released highly. When the cell necrosis or damaged the HMGB1 of the nucleus can be released into the extracellular under the role of TNF, triggered monocyte-macrophage cells to secrete pro-inflammatory cytokines,while pro-inflammatory cytokines can also further promote the secretion of HMGB1,so a positive feedback loop was formed.In the latter part of inflammatory reaction,this positive feedback has an important role in the maintenance of inflammatory response such as SLE.HMGB1 and B box protein were added in cultured macrophages respectively, it was shown that after stimulated by both the levels of TNF-a generation positively increased, after the use of anti-B box protein antibody results showed that both TNF-a in generate levels were significantly lower, all was told us B box domain plays major role in inflammation-causing effects of HMGB1 and it alone can play. Similarly, in macrophages, after adding A box protein the production of TNF-a levels under HMGB1 or B box protein stimulated can significantly reduced. So we can say A box protein plays an important role in inhibiting inflammation reaction and has become an effective antagonist of HMGB1.To explore the therapeutic effect of HMGB1 A box in the SLE and other autoimmune diseases, we carried out the expression vector construction, recombinant protein purification and biological activity studies.Methods and results1. Cloning and analysis of human high mobility group box 1 A boxWe optimized the coding sequence of mature human HMGB1 according to the report of GenBank, adjust the codon adaptation index (CAI)makes the value of CAI is 0.95, increase the content of GC in coding sequences in order to extend the half-lives of mRNA, the secondary structure of mRNA also was removed, plasmid DNA pUC57-HMGB1 was synthetized. According to the optimized sequence of HMGB1 one pair of primers was designed. restriction sites of Ndel and Xhol were added to the upstream of forward and reverse primers respectively, amplified human HMGB1-A box fragment with Taq DNA polymerase. PCR product was separated by agarose gel electrophoresis, then gel extraction kit isolated purpose gene fragments. The purified PCR product was ligated with pMD19-T vector and then transformed to the competent of E.coilDH5α. The positive clones were primary screened with colony PCR and restriction map analysis. Using reverse and forward primers the positive clones were sequenced with ABI3100 DNA sequencer. The results showed that the cloned A box was similar to optimized sequence. Therefore, the recombinant HMGB 1 A box vector, pMD19-T/HMGB 1 A box, has been successful constructed.2. Expression of High mobility group boxl A box in E.coli and identification of recombinant proteinTo generate the recombinant prokaryotic expression vector, pMD19-T/HMGB 1 A box were digested with two restriction enzymes Nde1 and Xhol. The purpose fragment was separated with low-melting agrose gel electrophoresis and inserted into pQE-T7-2 that was digested with the same enzymes. The recombinant was transformed into E. coli DH5a and inoculated LB plate with 50ug/mL kanamycin, positive recombinants were selected with colony PCR. Positive recombinant colonies were selected and inoculated into LB liquid medium that contain 50ug/ml kanamycin,then we extracted the plasmid and transformed to the competent of E.coilBL21. selection of a single colony was inoculated into LB liquid medium that contain 50ug/ml kanamycin. The bacterium harboring pQE-HMGB 1 A box was grown to early log phase in LB medium containing kanamycin (50ug/mL) at 37℃, expression of the recombinant HMGB 1 A box was induced with IPTG at 37℃for 4 hours. The cells were collected by centrifugation and analysised by SDS-PAGE和Western blotting. The SDS-PAGE of the bacterium lysate shown that there was an additional band that seemed to be about 14 KDa identified with previously reported that recombinant HMGB 1 A box expressed in E.coli. Using Western blotting analysis, recombinant protein can responses with anti-HMGB 1 polyclonal antibody and anti His-tag polyclonal antibody shown that recombinant protein is the specific human HMGB1 A box protein.3. Human HMGB1 A box protein purification and biological activity assayCollecting biomass then lyased with supersonic wave in the ice bath, supernatant was collected. Precipitated which was washed by 0.5% triton X-100 shall be crude inclusion bodies. The products of supernatant after supersonic wave and inclusion body lysate were analysis by SDS-PAGE. The results showed that recombinant protein present in the supernatant, suggesting that HMGB1 A box with soluble forms expressed in the cytoplasm of strain. The purpose protein was purified by Ni2+-NTA chromatography then analysised by SDS-PAGE. It was showed that the purity of recombinant protein HMGB1 A box was over 90%.Immune complex (IC) was prepared with serum of patients with SLE and Cell necrosis fluid which was prepared according to literature in the ratio of 1:25. Experiment was done under two groups:U937cell+IC group, U937cell+IC+HMGB1 A box group, incubate at 37℃for 24h under the condition of 50ml/L CO2. Total RNA was isolated, the levels of IFN-γ、BAFF and TNF-a were detected by RT-PCR while GAPDH as an internal reference. Agarose gel electrophoresis of PCR products were scanned by gel image scanner and analyzed using the relative index which can reflect the variation of expression of BAFF, IFN-y and TNF-a. The results showed that HMGB1 A box protein which could effectively inhibit the secretion of mononuclear cells in BAFF, TNF-a and IFN-y have significant biological activity.Conclusions1) Using recombinant DNA technique, we have successfully cloned human mature HMGB1 A box gene, and its DNA sequence was the same with the optimized sequence.2) The recombinant prokaryotic expression vector pQE-HMGB1 Abox containing human HMGB1 A box has been constructed and can express in the stable manner.3) Some methods involved in the recombinant HMGB1 Abox expression and preparation and purification have investigated and optioned.4) The recombinant protein is the specific human HMGB1 A box protein which accounted for 40% of the total bacterial protein,5) The recombinant protein have significant biological activity.更多还原