【作者】 吴忠祖；

【导师】 熊燕；

【作者基本信息】 中南大学 ， 药理学， 2007， 硕士

【摘要】 研究背景和目的心血管疾病是严重危害人类健康的常见病。一氧化氮介导的内皮依赖性血管舒张反应降低是大多数心血管疾病的早期病理表现。大量研究表明,内源性一氧化氮合酶(NOS)抑制物—非对称性二甲基精氨酸(ADMA)是导致血管内皮功能不全的重要物质。二甲基精氨酸二甲胺水解酶(DDAH)是内源性ADMA的主要代谢酶。已知DDAH存在两种亚型——DDAH1和DDAH2;前者主要分布于表达神经型NOS的组织,后者则主要分布于表达内皮型NOS的心血管组织;因此,DDAH2是决定心血管系统ADMA含量的关键因素。但目前尚无直接增加DDAH2表达和活性的药物。近年来发现一种来源于人类免疫缺陷病毒HIV-1 Tat(Trans-activator transcription)蛋白的蛋白功能区,称之为PTD区段(Protein transduction domain),能有效地引导肽段或蛋白质进入细胞,具有蛋白传送功能。因此,本课题将Tat PTD区段融合于人DDAH2蛋白的N端,使DDAH2蛋白具备穿膜能力,定位于细胞内并发挥作用。在此基础上,用多种心血管的危险因子如高糖、同型半胱氨酸、游离脂肪酸如棕榈酸处理培养的人脐静脉内皮细胞系(HUVEC-12),观察上述因素对细胞内DDAH1和DDAH2表达及DDAH活性的影响;并进一步探讨外源性给予Tat-hDDAH2蛋白能否直接增加细胞中DDAH2蛋白含量,抵抗上述因素对DDAH活性的抑制,从而为大规模开发可应用的重组蛋白产品奠定技术基础,为心血管疾病防治拓开新途径。另一方面,用重组的DDAH2融合蛋白作为抗原,制备家兔来源的抗血清,用于DDAH2表达的检测。方法①构建重组pGEX-6p-1-Tat-hDDAH2原核表达载体:以我室以前构建pcDNA3.1-hDDAH2质粒为模板,用Tat-hDDAH2上下游引物扩增Tat-hDDAH2基因,重组到T载体上,构建pGEM-Tat-hDDAH2质粒,然后再分别用SalⅠ和BamHⅠ双酶切pGEM-Tat-hDDAH2和原核表达载体pGEX-6p-1,连接重组成pGEX-6p-1-Tat-hDDAH2载体;再转化到宿主大肠杆菌BL21体内诱导表达,并通过优化诱导条件提高目的基因的原核表达水平和可溶性融合蛋白的含量。②重组Tat-hDDAH蛋白的纯化:将包涵体形式的重组蛋白溶于8mol/L的尿素溶液中,然后经过复性、透析得到复性蛋白GST-Tat-hDDAH;再将从细菌裂解液上清中的可溶性GST-Tat-hDDAH蛋白和从包涵体中得到的复性蛋白与还原型谷胱甘肽琼脂糖珠(Glutathione Sepharose 4B)充分混匀,经过Glutathione Sepharose4B的亲和吸附得到纯化的GST-Tat-hDDAH2融合蛋白,最后经过PreScission蛋白酶切,获得重组的Tat-hDDAH2蛋白。③抗hDDAH2抗血清的制备:重组pGEX-6p-1-hDDAH2载体;再转化到宿主细菌体内进行诱导表达,得到大量含GST-hDDAH2蛋白的包涵体;经2%的脱氧胆酸钠溶液洗涤后得到较纯化的包涵体,并将其作为抗原与佐剂充分乳化后经皮下注射免疫新西兰大白兔,制备抗DDAH2的多克隆抗体,并用ELISA、western blot检测DDAH2抗体的滴度和特异性,用于后续实验中DDAH2蛋白表达的检测。④重组Tat-hDDAH2蛋白的功能实验:培养人脐静脉内皮细胞系HUVEC-12,分别用心血管疾病的危险因子高糖、同型半胱氨酸、游离脂肪酸如棕榈酸(palmitic acid,PA)孵育HUVEC-12 16～48h,检测这些危险因子对内皮细胞DDAH1和DDAH2蛋白表达和DDAH活性的影响;并观察Tat-hDDAH2蛋白能否逆转上述因素对DDAH活性的损害。结果①经PCR、酶切及测序鉴定,原核表达载体pGEX-6p-1-Tat-hDDAH2(Fig.1～3)和pGEX-6p-1-hDDAH2(Fig.4)构建成功;并转化BL21大肠杆菌诱导表达,通过筛选不同的诱导条件,发现用较低的IPTG浓度(0.05 mM)在低温(25℃)环境下和适当地延长诱导时间(8 h)有利于可溶性融合蛋白GST-Tat-hDDAH2(Fig.5～7)和GST-hDDAH2(Fig.9～11)在细菌体内的表达,但在原核细胞中仍以不溶性的包涵体为主;将包涵体变性、复性后,再经纯化和PreScission蛋白酶酶切,最后获得重组的Tat-hDDAH2(Fig.8)和hDDAH2蛋白(Fig.12)。②将1升含有pGEX-6p-1-Tat-hDDAH2质粒和pGEX-6p-1-hDDAH2质粒的细菌培养液于12000 rpm 4℃离心10 min,去上清,将细菌用100 mlPBS重悬;经超声裂解后于12000 rpm 4℃离心10 min,分离上清中的可溶性融合蛋白与包涵体;将包涵体变性、复性后,分别将包涵体中复性的融合蛋白和上清中可溶性的融合蛋白经Glutathione Sepharose 4B亲和吸附,得到纯化的GST-Tat-hDDAH2融合蛋白2.169 mg和纯化的GST-hDDAH2融合蛋白2.571 mg(Tab.1～2);再分别将GST-Tat-hDDAH2和GST-hDDAH2融合蛋白经PreScission蛋白酶酶切后,可得到总量为1.362 mg的Tat-hDDAH2重组蛋白和1.469 mg的DDAH2重组蛋白(Tab.1～2)。③用含GST-hDDAH2蛋白的包涵体免疫新西兰兔,10 w后用乌拉坦(0.8 g/kg,i.v.)麻醉,经颈动脉插管取血,3000 rpm 4℃离心10 min分离血清,加入终浓度为0.02%的叠氮钠,从而获得特异性抗hDDAH2、效价达1:10000以上的多克隆抗体(Fig.13～14),分装后于-80℃保存,用于本课题中重组DDAH2蛋白、重组Tat-hDDAH2蛋白的检测以及内皮细胞中的DDAH2蛋白表达测定。④分别用0.1～1μM的Tat-hDDAH2蛋白孵育内皮细胞60 min,检测内皮细胞中DDAH2的表达及DDAH活性,以了解Tat-hDDAH2蛋白作用的量-效关系,结果发现,Tat-hDDAH2蛋白呈剂量依赖性地上调DDAH2表达,增加DDAH活性(Fig.15);再分别用1μM的Tat-hDDAH2蛋白孵育内皮细胞5～60 min,以观测Tat-hDDAH2蛋白作用的时-效关系,同样发现,Tat-hDDAH2蛋白呈时间依赖性地上调DDAH2表达,增加DDAH活性(Fig.16)。此外,用10～30mM的葡萄糖孵育内皮细胞48 h,以及用1～3 mM的同型半胱氨酸和0.1～0.5 mM的棕榈酸孵育内皮细胞16 h,均能呈剂量依赖性地抑制DDAH2表达(Fig.17,19),降低DDAH活性(Fig.18,20);用0.1～0.5μM的Tat-hDDAH2蛋白预孵育内皮细胞1 h,能逆转上述损伤因素对DDAH2表达和DDAH活性的抑制(Fig.17～20)。结论①利用分子重组技术,成功地构建了pGEX-6p-1-Tat-hDDAH2和pGEX-6p-1-hDDAH2原核表达载体;②分别将pGEX-6p-1-Tat-hDDAH2和pGEX-6p-1-hDDAH2载体转化BL21大肠杆菌,经纯化后获得了较高纯度的Tat-hDDAH2和hDDAH2重组蛋白,实验证明,重组的Tat-hDDAH2具有强的穿膜能力,并呈剂量和时间依赖性地增加内皮细胞中DDAH2的蛋白含量,上调DDAH活性,具有良好的功能;③利用重组的GST-hDDAH2包涵体蛋白免疫新西兰大白兔,成功地制备了高效价的抗hDDAH2的多克隆抗体,并能特异性地检测重组的GST-hDDAH2、Tat-hDDAH2和hDDAH2以及人脐静脉内皮细胞表达的DDAH2蛋白;④本研究揭示,高糖、同型半胱氨酸和游离脂肪酸如棕榈酸等心血管危险因子抑制内皮细胞DDAH活性主要与下调DDAH2表达有关;重组的Tat-hDDAH2蛋白能呈剂量依赖性地逆转上述损伤因子对人脐静脉内皮细胞DDAH2表达和DDAH活性的抑制。更多还原

【Abstract】 BACKGROUND Cardiovascular diseases,as the most common diseases of human being,have become one of the leading causes of death in developed countries as well as in many developing countries.Endothelial dysfunction, mainly characterized as reduction of nitric oxide(NO)-mediated endothelium-dependent vasodilatation,tends to precede diversed clinical manifestations of most cardiovascular diseases and to be an early pathological event.A substantial body of evidence suggests that elevated levels of endogenous nitric oxide synthase(NOS)inhibitor asymmetric dimethylarginine(ADMA)is involved in the mechanisms of endothelial dysfunction for cardiovascular diseases.Contribution of elevated endogenous ADMA to endothelial dysfunction predominantly lies in the decreased expression or activity of dimethylarginine dimethylaminohydrolase 2 (DDAH2),which is ADMA’s main metabolic enzyme and extensively located in endothelial cells.DDAH is composed of 2 isoforms,DDAH1 and DDAH2. DDAH1 is typically found in tissues of expressing neuronal NOS,whereas DDAH2 predominates in cardiovascular tissues of expressing endothelial NOS. So DDAH2 play a pivotal role in modulating ADMA metabolism in cardiovascular system.However,no medicine has ever been found to directly increase expression and activity of DDAH2.Until recently,studies have identified a protein domain,namely protein transduction domain(PTD),from human immunodeficiency virus tans-activator transcription protein(HIV-1 Tat),which can efficiently conduct peptides or proteins into living cell,thus playing a role of protein transduction.The objectives of this study are firstly to fuse Tat PTD to the N-terminal of DDAH2 protein,so as to enable DDAH2 protein to possess the ability of go through cell membrane and produce effects inside cells.To go a step further,we will also study effects of cardiovascular risks such as high glucose,homocystein and palmitic acid on expressions of DDAH1 and DDAH2 protein and activities of DDAH in cultured human umbilical vein endothelial cells(HUVEC-12)pretreatmented with or without Tat-hDDAH2 protein.Our work will provide a potential therapeutic method of treating cardiovascular diseases with recombined protein products.Moreover, we will prepare the anti-serum from New Zealand rabbits against recombination of GST-hDDAH2 fusion protein for the detection of DDAH2 protein expression by western blotting in this study.METHODS①Construction of recombination prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2:Taken the plasmid pcDNA3.1-hDDAH2 as template,we amplified Tat-hDDAH2 gene using the forward and reverse primers of Tat-hDDAH2,and ligated it with T-vector to construct pGEM-Tat-hDDAH2 plasmid.The plasmid and prokaryotic expression vector pGEX-6p-1 were digested with SalⅠand BamHⅠfor over night,respectively. We ligated the incised plasmid and vector to recombinate the vector of pGEX-6p-1-Tat-hDDAH2,which were then transformed into host bacteria E coli BL21 to induce maximal expression of the targeted gene and produce soluble fusion proteins in optimized conditions.②Purification of Tat-hDDAH recombination protein:First,recombinant proteins in form of inclusion bodies were dissolved in urea solution(8 M)and then after renaturation and dialysis, the renaturated protein GST-Tat-hDDAH was achieved.Nextly,the soluble GST-Tat-hDDAH protein in the supernatant of the bacterial lysate and the renaturated protein collected from the inclusion bodies were thoroughly mixed with Glutathione Sepharose 4B and then get purified by the affinity adsorption. Finally,the purified GST-Tat-hDDAH2 protein was incised by PreScission protease,and recombinant protein Tat-hDDAH2 was obtained.③Preparation of anti-hDDAH2 anti-serum:After recombination and transformation of pGEX-6p-1-hDDAH2 vector,the host bacteria were induced to express and produce a great quantity of inclusion bodies containing GST-hDDAH2 protein.The inclusion bodies washed by 2%sodium deoxycholate solution was thoroughly emulsified with the adjuvant and used to immunize New Zealand rabbits by subcutaneous injections.After the anti-DDAH2 polyclonal antibody was prepared,its titer and specificity were determined by ELISA and western blotting,respectively.We used it to detect expression of DDAH2 protein in the present study.④Functional study of Tat-hDDAH2 recombination protein:The human umbilical venous endothelial cell line HUVEC-12 were cultured and incubated with different risk factors of cardiovascular diseases,including high glucose,homocysteic acid and free fatty acid such as palmitic acid(PA)in the absence or presence of Tat-hDDAH2 protein for 16～48 h.Expressions of DDAH1 and DDAH2 proteins,as well as activities of DDAH,were detected to review the important role of DDAH2 in endothelial function and to explore the potential protective effects of Tat-hDDAH2 protein against these risk factors.RESULTS①As identified by PCR,digestion and sequencing,prokaryotic expression vectors of both pGEX-6p-1-Tat-hDDAH2(Fig.1～3)and pGEX-6p-1-hDDAH2(Fig.4)were successfully constructed and then transformed to E.coli BL21,in which were efficiently amplified to express soluble fusion proteins of GST-Tat-hDDAH2(Fig.5～7)and GST-hDDAH2 (Fig.9～11)by using a low concentration of IPTG(0.05 mM),at low temperature(25℃)and for a extended cultural time(8 h).However,in prokaryotic cells,the amplified fusion proteins occur in the form of insoluble inclusion bodies predominantly.So the inclusion bodies were denaturated, renaturated,purified and incised by PreScission protease to obtain the Tat-hDDAH2(Fig.8)and DDAH2(Fig.12)recombination proteins finally.②One liter of bacterial culture solution containing pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-DDAH2 plasmids was centrifuged at 12000 rpm,4℃,for 10 min.The supernatant was discarded and the pellet was resuspended in 100 ml PBS.After spallation by ultrasound,the resuspension solution was centrifuged at 12000 rpm,4℃for 10 min to separate the soluble protein in supernatant and insoluble inclusion bodies.After the inclusion bodies were denaturated, renaturated,the renaturated protein collected from the inclusion bodies and the soluble fusion protein in the supernatant were purified by affinity adsorption with Glutathione Sepharose 4B to get 1.545 mg of purified GST-Tat-hDDAH2 protein and 1.735 mg of GST-hDDAH2 fusion protein(Tab.1～2).These proteins were incised by PreScission protease to obtain 1.085 mg of recombinant Tat-hDDAH2 protein and 1.154 mg of recombinant hDDAH2 protein(Tab.1～2).③After immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2 for 10 weeks,we obtained the polyclonal antibody against human DDAH2 protein in a titer of 1:10000 by centrifuging the blood from the rabbits.In addtion,we used this anti-serum for measurement of recombinant DDAH2 protein,recombinant Tat-hDDAH2 proteins and expression of DDAH2 protein in HUVEC-12.④Incubation of HUVEC-12 with 0.1～1μM Tat-hDDAH2 for 60 min,the expression of DDAH2 and activity of DDAH in the HUVEC-12 were examined to see the dose-response of Tat-hDDAH2.The results showed that Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a dose-dependent manner(Fig.15).Moreover,the time course for Tat-hDDAH2 action was checked by treatment of endothelial cells with 1μM Tat-hDDAH2 for 5～60 min.Similiarly,Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a time-dependent manner.However,incubation of endothelial cells with 10～30 mM glucose for 48 h or with 1～3 mM Hcy and 0.1～0.5 mM PA for 16 h,not only DDAH2 expression was suppressed(Fig.17,19)but also DDAH activity was inhibited (Fig.18,20)in a dose-dependent manner.Pretreatment with 0.1～0.5μM Tat-hDDAH2 for 1 h could protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity.CONCLUSIONS①The construction of prokaryotic expression vectors pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was achieved.②The prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was transformed into Escherichia coli BL21.After purification,GST-hDDAH2 and GST-Tat-hDDAH2 recombination proteins in high purity were obtained.Further experiments confirmed that Tat-hDDAH2 protein can not only cross biological membranes efficiently but also prossess favourable function of up-regulating the DDAH2 expression and DDAH activity in endothelial cell in time- and dose-dependent manner.③Polyclonal antibody agaist hDDAH2 with a high potency was prepared successfully by immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2.This antibody can be used to detect recombination GST-hDDAH2,Tat-hDDAH2 hDDAH2 protein and DDAH2 expressing in human umbilical vein endothelial cells.④This study showed that reductions of DDAH activity induced by cardiovascular risk factors such as high glucose, homocystein and free fatty acids attributed to down-regulation of expression of DDAH2.Tat-hDDAH2 can protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity in in time- and dose-dependent manner.更多还原