人白细胞分化抗原CD59基因克隆及序列分析

为了获得人ＣＤ５９ｃＤＮＡ完整序列,以建立表达人ＣＤ５９分子的小鼠Ｔ细胞模型,更深入地探补体ＭＡＣ及ＣＤ５９分子与Ｔ细胞活化的关系。方法通过设计和合成成引物,提取细胞总ＲＡＮ进行逆转录反应,经ＰＣＲ扩增目的片段,并将基克隆于ＰＵＣ１８及ＰＵＣ１９质粒上,测定其ＤＮＡ序列。     

跨膜型CD59分子的构建及其在小鼠NIH3T3和EL-4细胞表达的研究

应用ＲＴ－ＰＣＲ方法,从Ｕ９３７细胞总ＲＮＡ中扩增得到编码人ＣＤ４６分子跨膜区和膜内区ｃＤＮＡ片段,用ＰＣＲ方法扩增得到编码成熟的ＣＤ５９胞外区蛋白的ｃＤＮＡ片段,连接并构建了跨膜型的ＣＤ５９分子ｃＤＮＡ。快速克隆于ｐＧＥＭ－Ｔ Ｅａｓｙ载体进行序列测定,证实了其阅读框的完整和序列的正确性。     

人CD38抗原胞外段基因克隆及表达

目的 克隆并表达人CD38抗原分子的胞外估基因。方法 采用RT－PCR法，从高表达CD38抗原的Daudi细胞系中，扩增CD38全长cDNA，并将其插入pGEM-T载体中。重新设计引物，从重组pGEMT载体中，扩增CD38抗体分子的胞外段基因，再亚克隆到表达载体pET28a( )，转化大肠杆菌BL21，用IPTG诱导表达。结果 经酶切鉴定及序列分析表明，克隆的CD38外段基因的序列与文献^[1,2]的报道完全一致。将该片段亚克隆到表达载体pET28a( ) ,经IPTG诱导在大肠杆菌BL21中获得表达。结论 获得了人CD38抗原分子胞外段基因及其原核表达产物，对进一步制备单克隆抗体，研究CD38分子的功能具有重要的意义。     

Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement.

We have examined the role of the human CD59 antigen in inhibiting complement-mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59-transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells.     

Functional activity of the membrane-associated complement inhibitor CD59 in a pig-to-human in vitro model for hyperacute xenograft rejection.

Hyperacute rejection triggered by activation of the recipient's complement system represents the major barrier to successful xenotransplantation. Transfer of human membrane-associated complement regulators to donor organs has been suggested as one strategy to interfere with complement-mediated hyperacute xenograft rejection. Pigs are discussed as potential organ donors. We therefore investigated a putative protective function of the membrane-bound complement inhibitor CD59 in a pig-to-human in vitro model of hyperacute xenograft rejection. Aortic porcine endothelial cells were transfected with human CD59 cDNA. Expression of human CD59 was demonstrated by cytofluorimetric and RNA analysis. Removal of CD59 from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) demonstrated its production as a glycosyl phosphatidylinositol (GPI)-anchored protein. Functional activity of the transfected CD59 was tested by a lactate dehydrogenase (LDH) release assay for complement-mediated lysis. Porcine endothelial cells expressing human CD59 were significantly protected from lysis by human serum complement compared with CD59- cells. The protective effect was abolished by preincubating the cells with anti-CD59 antibodies or PI-PLC. We calculated by Scatchard analysis that the established CD59+ cell line expressed a CD59 level comparable to that of human endothelial cells. Our results recommend the production of pigs transgenic for CD59.     

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59

CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required.     

人CD34抗原基因克隆及鉴定

ＣＤ３４抗原是一个阶段特异性白细胞分化抗原，在早期造血调控方面起着重要的作用，本文采用ＲＴ－ＰＣＲ方法，从高效表达ＣＤ３４抗原的ＫＧ－ｌａ细胞系中成功地克隆出ＣＤ３４抗原全长ｃＤＮＡ，并对重组基因进行了酶切鉴定和序列分析，结果表明除胞内区有一碱基改变，其它序列与报道完全一致。     

CD46 RNAi对CD59介导的T细胞信号转导的作用研究

目的构建携带针对CD46基因的pSUPER retro RNAi逆转录病毒载体,研究糖基磷脂酰肌醇(GPI)锚定蛋白CD59与CD46在介导T细胞信号转导中的相关性。方法将能转录产生靶向CD46小发夹RNA(shRNA)的寡核苷酸序列,克隆入逆转录病毒载体pSUPER retro,转化大肠杆菌JM109并转染Jurkat细胞。将Jurkat细胞分为未转染的Jurkat细胞组(Ⅰ组)、转染空质粒的Jurkat细胞组(Ⅱ组)、转染CD59干扰质粒的Jurkat细胞组(Ⅲ组)及转染CD46干扰质粒的Jurkat细胞组(Ⅳ组)。用RT-PCR、Western blot技术检测各组细胞中的CD59、CD46基因的表达水平。用噻唑蓝(MTT)比色法检测CD46与CD59联合作用对4组Jurkat细胞的增殖效应。结果重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序,结果表明序列正确,构建成功,稳定转染后,Ⅳ组细胞CD46分子的表达被成功抑制,Ⅲ组细胞CD59分子的表达被抑制。Ⅰ组和Ⅱ组细胞CD46与CD59单抗联合作用后,增殖能力明显高于Ⅲ组、Ⅳ组(P<0.05);但Ⅰ组和Ⅱ组,Ⅲ组和Ⅳ组之间无差异。结论 CD59可增强CD46对T细胞信号转导的效应。     

重组跨膜型人CD55分子在小鼠NIH3T3细胞上的表达及其补体溶破保护功能的研究

目的 :在小鼠NIH3T3细胞转染表达人天然GPI锚固型CD5 5和重组跨膜型CD5 5 TM分子 ,观察比较它们对人补体溶破异源细胞的抑制功能。方法 :将带有CD5 5cDNA、CD5 5 TMcDNA的重组逆病毒表达质粒CD5 5 pLXSN、CD5 5TM pLXSN经脂质体法转染PA317细胞 ,用病毒上清感染小鼠成纤维母细胞NIH3T3。经G418加压筛选 ,利用FACS检测获得表达CD5 5和CD5 5 TM分子的阳性细胞克隆 ,通过MTT比色法比较两种分子对人血清补体溶破细胞的抑制功能有无差别。结果 :细胞转染筛选获得多个表达跨膜型人CD5 5分子的NIH3T3细胞克隆 ,补体杀伤试验证实其具有抑制人补体溶破的功能 ,且两种分子的补体抑制功能无明显差异。结论 :成功地建立了稳定表达天然CD5 5、跨膜型CD5 5分子的小鼠NIH3T3细胞 ,证实其表达的GPI型CD5 5分子和CD5 5TM分子均具有抑制人补体溶破细胞的功能 ,为进一步探讨应用跨膜型的CD5 5分子对PNH进行基因治疗奠定了基础。     

人补体膜辅助调节蛋白MCP的基因克隆及其真核表达的研究

目的　克隆获得编码人补体膜辅助调节蛋白（ ＭＣＰ） 的ｃＤＮＡ,并对其在真核细胞的表达及功能进行研究。方法　应用ＲＴＰＣＲ 方法,从Ｕ９３７ 细胞总ＲＮＡ 中扩增编码人ＭＣＰ 分子的ｃＤＮＡ片段, 快速克隆于ｐＧＥＭＴＥａｓｙ 载体,测定其序列。将该片段重组于ｐＬＸＳＮ 载体,电穿孔转染ＮＩＨ３Ｔ３ 细胞,经ＦＡＣＳ 检测筛选表达ＭＣＰ 的阳性细胞克隆,用补体溶破试验鉴定其抑制人补体溶破的功能。结果　ＲＴＰＣＲ 扩增得到１ １４４ｂｐ 的编码人ＭＣＰ 分子的ｃＤＮＡ 片段,序列分析表明该ｃＤＮＡ编码的蛋白为ＳＴＣ＋ ＣＹＴ２ 亚型。细胞转染筛选获得多个表达ＭＣＰ 的ＮＩＨ３Ｔ３ 阳性细胞克隆,补体溶破试验证实其具有抑制人补体经典途径和旁路途径溶破的功能。结论　本研究为进一步探讨不同亚型结构的ＭＣＰ 分子与功能的关系及其应用奠定了基础。     

转录因子Sp1在肿瘤细胞CD59表达调控中的作用

目的:构建针对Sp1基因siRNA真核表达载体,转染前列腺癌细胞PC-3,研究反式作用因子Sp1对CD59表达的影响.方法:应用siRNA表达载体介导的RNAi技术,构建含特异性Sp1基因的重组载体pSUPER-siSp1,脂质体法转染前列腺癌细胞,G418筛选建立稳定表达转染基因的细胞株.RT-PCR和Western blot检测转染细胞中Sp1和CD59基因的表达,染料释放试验判断CD59基因抑制后对补体溶破的抵抗作用.结果:成功构建了Sp1基因siRNA真核表达载体.转染PC-3细胞可表达荧光蛋白,稳定转染的PC-3细胞CD59基因的mRNA和蛋白水平降低,染料释放实验表明CD59基因受抑制后对补体溶破的抵抗作用降低.结论:siRNA-Sp1重组载体有效地抑制了CD59的表达,降低CD59的抗补体活性,结果证明反式作用因子Sp1是CD59表达调控中重要的转录因子,为探讨CD59在肿瘤细胞中高表达的研究奠定了基础.     

Decreased expression of 20-kD homologous restriction factor (HRF20, CD59) on T lymphocytes in Epstein–Barr virus (EBV)-induced infectious mononucleosis

HRF20 (CD59) is one of the membrane-associated complement regulatory proteins. The characteristic function of CD59 is to prevent membrane attack complex (MAC) formation on the cell surface and to protect the cell from complement-mediated cell lysis. We examined the expression of CD59 antigen on T cell subpopulations in patients with acute infectious mononucleosis (IM) and analysed the relationship between the amount of CD59 expression and activation-induced cell death of mature T cells with apoptosis. Decreased expression of CD59 on CD8⁺ T cells, especially on CD45RO⁺ and HLA-DR⁺ activated T cells, was marked in acute IM patients. In contrast, activated CD4⁺ T cells from IM patients expressed as much CD59 antigen as CD4⁺ T cells from healthy volunteers. After incubation-induced cell death, viable CD8⁺ T cells showed normal amounts of CD59 antigen on their surface. CD59^dim CD8⁺ T cells were more susceptible to apoptosis than CD59^bright CD8⁺ T cells. These findings suggest that decreased expression of CD59 on CD8⁺ T cells may discriminate the susceptibility of activated CD8⁺ T cells to activation-induced cell death in IM.     

CD59锚定蛋白对CD55介导的T细胞信号转导的增强效应

目的:研究糖基磷脂酰肌醇(GPI)锚定蛋白CD59对CD55介导T细胞信号转导的增强效应。方法:实验分为未转染的Jurkat细胞组(Ⅰ组)、转染空质粒的Jurkat细胞组(Ⅱ组)及转染CD59干扰质粒的Jurkat细胞组(Ⅲ组)。用RT-PCR检测3组细胞中的CD59基因表达水平。用噻唑蓝(MTT)比色法、免疫印迹技术和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对3组Jurkat细胞的增殖效应,Src家族酪氨酸激酶(SrcPTK)磷酸化的水平及细胞内钙离子的变化。结果:稳定转染后,Ⅲ组细胞CD59分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力,SrcPTK磷酸化水平及钙离子浓度均明显高于Ⅲ组(P<0.05);但Ⅰ组和Ⅱ组之间无差异。结论:CD59可增强CD55对T细胞信号转导的效应。     

CD59-CD2对T细胞信号转导的作用

目的将构建的CD59-linker-CD2融合基因真核表达载体在Jurkat细胞中稳定表达,研究CD59与CD2分子在T细胞信号转导中的相互作用,探讨CD59向T细胞内传递信号的相关机制。方法酶切鉴定构建的pIRES-CD59-linker-CD2融合基因真核表达载体,脂质体法转染Jurkat细胞,G418筛选建立稳定转染细胞系,免疫酶法检测转染细胞表面CD59的表达,Western blot检测转染细胞中CD59蛋白的表达;用CD59单克隆抗体作用于各组Jurkat细胞,使用激光共聚焦显微镜检测细胞浆内的Ca2+,ELISA检测细胞上清中白介素2(IL-2)的变化,比较各组差异。结果经酶切鉴定证实携带CD59-linker-CD2融合基因的重组质粒扩增成功;免疫酶法和Western blot证实CD59在转染细胞中稳定表达,且转染组L-Jurkat细胞比正常组Ju-rkat细胞CD59表达量增加,二者比较其差别有统计学意义(P<0.05);与正常细胞相比,胞浆内Ca2+和IL-2在转染细胞中均高表达,两组细胞比较其差别有统计学意义(P<0.05)。结论 pIRES-CD59-linker-CD2融合基因真核表达载体可在Jurkat细胞中稳定表达,CD59mAb交联刺激后,CD59可通过CD2向细胞内传递信号,引起细胞内信号分子的一系列变化,为研究CD59与CD2在T细胞信号转导中的协同作用及进一步探讨CD59向T细胞内传递信号的机制奠定基础。     

人碱性成纤维细胞生长因子(bFGF)cDNA的克隆

为了研究人碱性成纤维细胞生长因子在治疗神经系统疾病中的作用 ,克隆其 c DN A序列 ,并添加 K ozak序列 ,以用于真核细胞表达。本实验提取我国自建的脑胶质瘤细胞系 BT32 5总 RN A,设计上、下游引物用逆转录 PCR法扩增 c DNA片段 ,然后重组于 p GEM- 3zf( + )载体 ,酶切鉴定插入片段正确后测序。结果 :RT- PCR扩增到 4 73bp的带有 K ozak序列的 c DN A片段。显示 :克隆到碱性成纤维细胞生长因子 c DN A片段 ,可用于真核细胞表达     

可溶性嵌合补体抑制分子的构建及表达

目的：制备能抑制补体活化的2个关键环节（C3/C5转化酶和MAC的形成）的一种新型、高效补体抑制剂。方法：先设计引物，然后通过PCR技术扩增重组可溶性CD46/CD55/CD59嵌合分子cDNA片段，重组于pSecTagA真核表达载体上，分别利用COS-7细胞和中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞进行表达，并用抗人CD46多克隆抗体及抗人CD55、CD59单克隆抗体对表达产物进行western bolting鉴定。结果：DNA测序证实，前端带有小鼠IgGκ链前导序列，后端融合有编码6个组氨酸碱基的CD46/CD55/CD59嵌合分子cDNA片段的阅读框完整。免疫印迹结果显示，表达的重组蛋白分别能与抗人CD46多克隆抗体和抗人CD55、CD59单克隆抗体结合。结论：成功构建并在真核细胞内表达了重组可溶性CD46/CD55/CD59嵌合分子，为进一步研制和开发新一代多功能、多靶点补体抑制剂奠定了基础。     

突变人CD59基因真核表达系统的构建

目的：构建2种突变人CD59(HMCD59)重组质粒，建立高效真核表达系统。方法：采用基因点突变技术在CD59易于糖基化的位点K41-H44将H44基因位置变更或增加K的数目，构建2种不同的CD59突变基因，各设计两条互补突变引物和两条常规引物，以已构建CD59 cDNA为模板，3重PCR定点诱变扩增突变基因，EcoR Ⅰ酶切重组人真核表达质粒载体pALTER-MAX，利用阳离子脂质体(Lipfectamine-2000)将重组质粒和pcDNA3共转染中国仓鼠卵巢细胞(Chinese hamster ovary，CHO)进行表达。结果：通过PCR定点诱变成功获得目的CD59突变基因，序列测定及酶切鉴定均证实成功构建了pALTERMAX-突变CD59重组真核表达载体系统，突变基因长度约500bp。阳离子脂质体(Lipfectamine-2000)将重组载体质粒和pcDNA3共转染导入真核受体细胞(CHO)，C418筛选出了稳定细胞克隆。结论：以突变CD59基因和pALTER-MAX质粒为基础构建的真核表达载体构建成功，脂质体转染法将重组质粒导入真核细胞并获得膜表面突变CD59分子的高效表达。     

前列腺细胞高表达的CD59分子活性位点的封闭研究

目的 观察噬菌体肽库筛选的短肽(SP22)对前列腺癌细胞高表达的正常或突变CD59分子活性位点的封闭效果.方法 将正常和突变CD59基因、pIRES空质粒分别转染PC-3细胞;流式细胞术榆测细胞表面CD59分子的表达;RT-PCR检测CD59基因的mRNA表达;补体溶解试验观察SP22埘补体介导的PC-3细胞溶解的影响.结果 CD59基因成功转染并表达;wPC-3细胞(转染正常CD59基因)和mPC-3细胞(转染突变CD59基因)内CD59mRNA表达水平显著高于pPC-3细胞(转染空质粒)(P＜0.01);SP22使3种细胞的溶解率明显提高(P＜0.01),但升高的模式和幅度显著不同.结论 SP22不能封闭突变CD59分子的补体结合位点,但可与PC-3细胞表面的正常CD59分子高效结合并中和其补体抑制活性,进而显著增强补体对PC-3细胞的溶解.     

siRNA特异沉默卵巢癌细胞CD59基因的裸鼠移植瘤研究

目的:通过体内动物实验研究CD59-siRNA对卵巢癌细胞CD59的沉默效应及抑瘤作用,探讨CD59在肿瘤免疫逃逸中的作用.方法:将转染CD59干扰质粒的A2780细胞(T组)、转染空质粒的A2780细胞(V组)及未转染的A2780细胞(C组)分别接种于裸鼠皮下,通过绘制肿瘤生长曲线,裸鼠移植瘤组织切片CD59 mRNA原位杂交及CD59蛋白的免疫组化研究其抑瘤效应及对CD59的沉默效应.结果:肿瘤生长曲线显示,与对照组相比,CD59干扰质粒转染组肿瘤生长明显受抑制(P<0.05).原位杂交及免疫组化结果表明,干扰组的CD59 mRNA及CD59蛋白与对照组相比显著降低(P<0.05).结论:裸鼠体内实验表明,特异性沉默CD59基因的siRNA表达载体可以明显抑制CD59的表达,增加了卵巢癌细胞对补体攻击的敏感性,从而抑制卵巢癌在体内的生长,进一步说明了CD59在肿瘤免疫逃逸中的作用.     

Expression and function of CD59 on colonic adenocarcinoma cells

The expression and function of CD59, a 19–25 kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement, was analyzed on normal and malignant human colonic epithelial cells. Analysis by immuno-fluorescence demonstrated a weak apical expression of CD59 on normal intestinal epithelium, with an increased expression on adenocarcinoma cells. The expression of CD59 was greatest on tumor cells with poor differentiation. The functional activity of CD59 on human adenocarcinoma cells was investigated using the colonic adenocarcinoma cell line HT29, CD59 on HT29 cells was glycosyl-phosphatidylinositol-linked, and had a molecular mass of 19–25 kDa. HT29 cells expressed approximately four times more CD59 than leukocytes, and showed a high resistance to antibody-dependent complement-mediated lysis. Blocking of CD59 with divalent antigen-binding F(ab′)₂ fragments of the anti-CD59 monoclonal antibody 1F5 resulted in a dose-dependent increase in complement-mediated lysis, suggesting that CD59 may be of importance in protecting colonic adenocarcinoma cells against complement-mediated cytolysis.