基于人DAF框架的酵母展示肽库的构建与鉴定

以hDAFcDNA为模板 ,用随机引物分别通过大引物PCR和交叠PCR方法引入突变 ,酶切后克隆到酵母表面展示载体pYD1 ,构建DAF pYD1重组质粒 ,转化酵母细胞 ,PCR鉴定随机性 ,流式细胞仪分析DAF的表达。结果表明 ,随机性达到理论要求 ,通过流式细胞仪检测到酵母细胞表面有DAF分子的表达。以上结果表明 ,成功构建了基于DAF框架的酵母表面展示肽库 ,为进一步筛选DAF的突变体库奠定了基础。     

补体激活调节因子作为补体抑制剂的研究进展

补体激活调节因子 (RCA)家族的补体调节蛋白可阻断人类疾病时补体的不适当激活。它们均作用于补体前端反应 ,通过其短同源重复 (SCR)与C3b或C4b结合 ,或促进C3和C5转化酶的衰变 ,或辅助Ⅰ因子裂解C3b和C4b ,从而达到抑制补体激活的目的。本文对近年来RCA家族中部分成员作为补体抑制剂的研究进展作一综述。     

人杀菌通透性增强蛋白功能性N端片段在酵母细胞表面的展示和鉴定

目的：建立BPI23酵母细胞表面展示方法,使之表达具有生物活性的BPI23蛋白。方法：构建pYD1-BPI600重组质粒,转化酵母细胞,经半乳糖诱导使BPI23在酵母细胞表面表达;采用间接免疫荧光法,用荧光显微镜和流式细胞仪检测BPI23在酵母细胞表面的展示情况;采用鲎基质显色法,检测BPI23中和内毒素的生物学活性。结果：酶切和DNA测序鉴定证实,pYD1-BPI600重组质粒含有人BPI600基因片段。荧光显微镜和流式细胞仪检测结果显示,BPI23在pYD1-BPI600重组质粒转化的酵母细胞表面得到展示;在诱导后第24小时展示BPI23的酵母细胞数占总细胞数的39%。酵母细胞表面展示的BPI23具有中和内毒素的活性。结论：成功构建了pYD1-BPI600酵母表达载体,转化后在酵母细胞表面展示了功能性目的蛋白,为BPI23突变体库的建立和功能优化BPI23突变体的筛选奠定了基础。     

真核蛋白表面展示系统

酵母表面展示和杆状病毒表面展示是近年来发展起来的新的蛋白表面展示技术,可用于展示需糖基化作用、二硫键异构化等翻译后修饰才表现功能活性的复杂真核蛋白。本文简述了该技术的基本原理、应用、研究进展及其发展前景。     

衰变加速因子在非小细胞肺癌中的表达及其临床意义

目的 检测衰变加速因子（decay accelerating factor,DAF,CD55）在非小细胞肺癌（non-small cell lung carcinoma,NSCLC）中的表达,分析其表达与临床分期、化疗用药及预后的关系。方法 免疫组化法检测8例NSCLC癌旁组织和36例NSCLC手术标本中DAF的蛋白表达,分析临床资料。结果 免疫组化结果显示36例NSCLC标本中,共有18例（50．0％）表达DAF,其中18例肺鳞癌中有15例（83．3％）表达,而16例肺腺癌中有3例（18．8％）表达,两者有显著差异（P〈0．05）;不同的病理分期的DAF免疫组化平均积分光密度表达有显著性差异（P〈0．05）;DAF的表达与无病生存期无相关关系（P〉0．05）。结论 NSCLC中有DAF的表达,尤其是肺鳞癌;提示在NSCLC抗体治疗中应同时阻断DAF的免疫抑制效应。     

伯氏疏螺旋体补体调节因子捕获表面蛋白介导其补体逃逸机制的研究进展

伯氏疏螺旋体补体调节因子捕获表面蛋白(complement regulator-acquiring surface protein,CRASP)通过捕获宿主补体调节因子,干扰补体激活机制,防止螺旋体在宿主体内定植过程中被活化的补体系统杀灭,有利于螺旋体的生存和造成慢性感染.全面了解CRASP与补体调节H因子(facto...     

补体系统与树突状细胞的相互关系

树突状细胞和补体系统是天然免疫的两个重要组成部分,两者有着极其紧密的联系。树突状细胞是补体产生的重要来源,其合成分泌的补体成分能调节自身的分化和发育,进而影响获得性免疫应答。     

人衰变加速因子的二级结构与B细胞表位预测

目的分析预测人衰变加速因子的二级结构特征及B细胞表位。方法比较Chou&Fasman经典方案和基于多重序列比较的PHDsec二级结构预测方案的预测准确率,应用较优方案对DAF的二级结构进行预测分析;运用Hopp&Woods的亲水性方案及PHDacc可及性方案预测DAF的亲水性和可及性,结合DAF的二级结构特征,分析预测DAF的B细胞表位。结果PHDsec方案对SCR的预测准确率明显高于Chou&Fasman方案,DAF的SCR1-4中无α螺旋,仅包含β折叠及袢;推测最可能的抗原表位位于Pro73-Val79、Arg130-Leu139及Glu156-Cys163;SCR1、SCR2与SCR3、SCR4在亲水性及二级结构分布方面具有较大差异。结论研究的分析预测结果将有助于确定DAF的B细胞表位及其生物学活性部位。     

衰变加速因子介导Jurkat细胞的PTK信号传递机制的实验研究

为研究衰变加速因子 (DAF )介导Jurkat细胞信号传递的机制 ,本文应用免疫印迹及化学发光技术 (ECL)发现交联DAF单抗可使CD3+ 与CD3 Jurkat细胞总蛋白酪氨酸磷酸化水平增加 ,但磷酸化水平不同。免疫共沉淀实验检测到DAF与Src家族酪氨酸激酶 (SrcPTK )可发生共沉淀反应。去污剂不溶膜复合物 (DIG )抽提与鉴定检测到DIG内有SrcPTK与DAF的特异性聚集。说明DAF对Jurkat细胞的信号传递有辅助激活效应 ,此效应可能是通过其以与膜微区相关的所联系的SrcPTK实现的     

Depletion of complement does not impact initiation of xenobiotic-induced autoimmune disease

Deficiency in Daf1, a complement regulatory protein, has been shown to exacerbate development of various autoimmune diseases and recent studies have suggested that this may be explained by Daf1 acting to limit T-cell hyper-responsiveness. It has been suggested that the absence of Daf1 aggravates autoimmune disease in a complement-dependent manner, but others have shown that activation of T cells in the absence of Daf1 can be complement independent. However, the relationship between Daf1, complement components, lymphocyte activation, cytokine expression and antibody production remains to be determined in mice that are not Daf1 deficient. We have recently demonstrated, in murine mercury-induced autoimmunity (mHgIA), that an accumulation of CD44(high) Daf(low) CD4(+) T cells is associated with the development of autoimmunity. In this study we observed that complement depletion does not affect the accumulation of activated CD4(+) T cells, elevation of splenic interleukin-4 expression and autoantibody production in mHgIA. In addition, neither the accumulation of CD44(high) Daf(low) CD4(+) T cells nor the down-regulation of Daf1 expression on CD4(+) T cells was influenced by a lack of complement. In conclusion, these studies show that initiating events in xenobiotic-induced autoimmunity, including lymphocyte activation, cytokine expression and autoantibody production, are not dependent on complement.     

Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF)

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.     

Deficient surface expression of glycosylphosphatidylinositol-anchored proteins in B cell lines established from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired type hemolytic disorder. Hematopoietic cells of patients with PNH are deficient in glycosylphosphatidylinositol (GPI) anchored membrane proteins. Since some membrane-bound complement inhibitors, such as CD59 and decay accelerating factor (DAF), are GPI anchored proteins, abnormal cells from patients with PNH are sensitive to complement attack. Their myeloid and erythroid cells are affected more than their lymphoid cells. Patients whose B cells were severely deficient in GPI anchored proteins were chosen to establish cell lines by Epstein-Barr virus mediated transformation. The lines established (SS-1-, TK-1-, and TK-14- cell lines) had the following characteristics of PNH. First, GPI anchored proteins were completely absent from the surface of SS-1- and TK-14- cells, and were expressed at very low levels on TK-1- cells, whereas polypeptide anchored proteins were normally expressed on these three lines. Secondly, DAF mRNAs of the SS-1- cell line were qualitatively and quantitatively indistinguishable from those of a control, wild-type cell line. Third, pro-CD59 and pro-DAF molecules were detected intracellularly in these cell lines, their pro-CD59 being smaller and more hydrophilic than that from a wild-type cell line. These cell lines should be useful in further studies on the pathogenesis of PNH.     

Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: identification of forms increased in cancer patients' sera

Normal human sera contained 10–60 ng/ml of soluble membranecofactor protein (MCP, CD46) whereas sera of >50% of thecancer patients contained >60 ng/ml. MCP purified by immunoaffinitychromatography from both normal and cancer patients' sera consistedof three bands of 56, 47 and 29 kDa on SDS-PAGE/immunoblotting.The upper two components were increased in cancer patient sera.The 56 and 47 kDa soluble forms served as a cofactor for factorI-mediated cleavage of C3b. MCP expressed on Chinese hamsterovary (CHO) cells protects host cells from human C3 depositionand complement-mediated cytolysis, especially by activationof the alternative pathway. In this same assay system, exogenouslyadded soluble MCP also protected untransfected CHO cells; however,its potency was much less than that of the endogenous membraneform. For example, 8 µg/ml of soluble MCP was equal to10⁴copies/cell of the expressed MCP. Recombinant soluble formspossessed similar activity to the naturally occurring solubleforms and high doses (>150µg) blocked Arthus-like reactioninduced in guinea-pigs by anti-Forssman antibody. These dataestablish that soluble forms of MCP are present in human serathat possess cofactor activity and their concentrations, especiallythe 56 and 47 kDa forms, are increased in sera of cancer patients.High doses of the recombinant soluble forms may be therapeuticallyuseful for suppressing inflammatory responses.     

Mitigating placental injuries through up-regulating DAF in experimental APS mice: new mechanism of progesterone

Anti-phospholipid syndrome (APS) is characterized by recurrent pathological pregnancy, arterial or venous thrombosis in the presence of anti-phospholipid antibody (aPL). Complement activation is recognized as an intermediate link leading to placental thrombosis and placental inflammation in APS model mice. Decay accelerating factor (DAF, CD55), MAC-inhibitory protein (MAC-IP, CD59) and membrane co-factor protein (MCP, CD46) are important complement inhibitory proteins (CIPs) highly expressed in normal placenta to curb excessive complement activation and its mediated injuries. Anti-β2 glycoprotein I (anti-β2GPI) antibody is an important aPL. We found that placental DAF and CD46 decreased in β2GPI passively immunized APS model mice, accompanied by C3 deposition, neutrophil infiltration and increased proinflammatory cytokine levels detected in its placenta. Progesterone supplement can up-regulate DAF but not CD46 expression, curb C3 activation and decrease proinflammatory cytokines levels to reduce fetal loss frequency. Progesterone receptor antagonist (mifepristone) or knock-down DAF with specific siRNA, above the protective effects of progesterone, were significantly weakened. Another sex hormone, oestrogen, has no significant effect on placental DAF and C3 contents and fetal loss frequency in the APS mice model. This may be an important mechanism by which progesterone induces maternal–fetal immune tolerance. At the same time, it may provide evidence for the use of progesterone in APS abortion patients.     

衰变加速因子在Jurkat细胞增殖及分泌IL—2中的作用

目的 研究衰变加速因子对CD3阳性与阴性Jurkat细胞的增殖效应及对IL－2分泌的影响。方法 应用MTT比色实验观察交联DAF单抗DG3与固相化抗CD3联合作用对CD3阳性与CD3阴性Jurkat细胞增殖效应;IL－2依赖CTLL^3H-TdR掺入实验检测CD3阳性与CD3阴性Jurkat细胞IL－2的分泌;细胞ELISA检测CD3阳性与CD3阴性Jurkar细胞IL－2R的表达。结果 交联DG3可协助促进固相化抗CD3对CD3阳性Jurkat细胞的增殖效应和IL－2分泌,并可引起IL－2Rα链表达增加,此作用可被Src家族酪酸激酶抑制剂PP2所抑制,而对CD3阴性Jurkat细胞无此作用。结论 衰变加速因子可协同促进固相化抗CD3对CD3阳性Jurkat细胞的刺激增殖效应、IL－2分泌及IL－2R表达增加。