【作者】 江海燕；

【导师】 富宁；

【作者基本信息】 第一军医大学 ， 免疫学， 2004， 硕士

【摘要】 可溶性CD14检测体系的建立及其应用研究 CD14是革兰氏阴性菌内毒素脂多糖(LPS)的高亲和力受体之一。除LPS外，CD14还介导肽聚糖(PGN)、脂磷壁酸(LTA)、脂蛋白等多种病原体成分与细胞的反应，它是天然免疫中重要的模式识别分子。CD14有两种存在形式：细胞膜表面的膜型受体(mCD14)和可溶性受体(sCD14)。sCD14存在于人体血液，脑脊液等体液中，在感染、创伤、烧伤和SLE等多种疾病中，sCD14的水平会升高，且与疾病的发生、发展相关。目前sCD14已成为机体单核／巨噬细胞活化的标志，用于监测疾病的发生和发展。 国内关于sCD14的研究极少，究其原因，与目前尚无国产试剂盒而国外进口试剂盒价格昂贵有关。为解决这一困难，推动该领域的研究，并为国内关于CD14的基础研究和临床研究提供技术支持，本研究建立了人sCD14单多抗双夹心ELISA检测体系；同时，还从人血浆中提取了天然sCD14，并进行了人CD14基因的克隆和表达，为在后续工作能深入研究CD14的生物学功能，探讨CD14与Toll样受体的相互作用以及在抗感染免疫中发挥的作用奠定了必要的基础。 一、建立人sCD14单多抗双夹心ELISA检测体系 首先进行了抗人CD14单克隆抗体的纯化和多克隆抗体的制备。抗人CD14单克隆抗体60bca由本实验室利用单抗细胞株HB-247按常规方法制备小鼠腹水，用HiTrap protein G亲和层析柱纯化后，获得单抗15mg。用转染人CD14基因的人单核细胞THP1-CD14免疫家兔，获得了兔抗人CD14多克隆抗体，该抗血清特异性好，能够满足本研究的需要。 利用已获得的抗人CD14单抗和多抗建立人sCD14单多抗双夹心ELISA检测体系。通过优化实验条件，确定了单抗60bca包被浓度，兔抗人CD14多抗和HRP-羊抗兔IgG的工作浓度；用IBL公司进口人sCD14检测试剂盒标定混合正常人血清，作为本检测体系的标准品和质控血清。 经过测试证明，本研究建立的sCD14检测体系各项技术指标达到国外进口试剂盒的技术水平，具体为:敏感性可达sng/ml，测量范围sng/m1一120ng/m1;特异性强;重复性好，批内重复测定变异系数为3.27%~9.72%，批间重复测定变异系数为6.7%一13.8%;准确性高，与国外进口试剂盒同时测量相同样品，所得结果相当。 本研究利用自行建立的sCD14检测体系，测定了154名正常人，137名神经内科住院患者以及300余名心内科住院患者血清标本。经过统计分析发现:健康人群中不同年龄组血清sCD14水平有一定差异;脑梗塞患者血清sCD14水平显著高于同年龄段正常对照组;急性冠脉综合征(急性心肌梗死和不稳定性心绞痛)患者血清sCD14水平也有显著升高。以上研究为进一步探讨sCD14在心脑血管疾病发生、发展中的作用机制提供了重要依据。二、人可溶性CD14的提取与制备 为获得纯化人CD14蛋白用于后续工作，我们利用免疫亲和层析技术从人血浆中提取天然sCD14蛋白。将纯化的单抗60bca与eNBr-aetived sePharose 4B交联，制备了sCD14亲和层析柱，从20oml人血浆中纯化获得400林9 sCD一4蛋白，经Westem Blot和商品ELIsA试剂盒鉴定，其特异性较好，保持了天然蛋白的生物活性。 尽管天然sCD14蛋白在构象和功能等方面都优于重组CD14蛋白，但人血浆中scD14含量较低，且来源有限，难以满足研究工作的需求。因此，我们还利用分子生物学技术进行了人CD14全长基因的克隆和表达。具体工作包括:(l)提取THPI一CD14细胞总RNA，通过RT一PCR扩增得到人CD14全长基因，测序鉴定证明本实验所获得的人CD14基因序列与Ge丘Bank中报道的一致;(2)利用己获得的CD14基因片断成功构建了人CD14原核表达载体pET28b/cD14和酵母表达载体ppICZaA/CD14。(3)用IPTG诱导重组人eD一4在大肠杆菌中表达，经SDS.pAGE和westem blot鉴定，cD14蛋白在大肠杆菌中获得了大量表达，但全部以包涵体形式存在，虽经过变性、复性处理，仍不能恢复CD14蛋白活性。(4)用甲醇诱导重组人CD14在Pichia酵母中表达，经SDS一AGE和westem blot鉴定，重组人cD14蛋白以可溶形式存在于酵母菌培养上清中，但双夹心ELISA测定其表达水平较低，表达量仅为48ng/ml。 我们的后续工作包括:(l)进一步推广SCD14检测体系在基础研究与临床中的应用;(2)继续探索获得大量表达重组人CD14蛋白的实验条件，并通过分析CD14的抗原表位，有选择地进行重组人CD14蛋白部分片断的表达，目前CD 14N末端1 52aa片段的表达已在进行中。更多还原

【Abstract】 CD14 is one of specific receptors for bacterial lipopolysaccharide (LPS) with high affinity. In addition to LPS, CD 14 has been reported to be receptors for a wide variety of bacterial products, such as peptidoglycan (PGN), lipoteichoic acid (LTA), lipoprotein (BLP), etc. Thus, CD 14 is an important component of the innate immune system and can be defined as pattern recognition receptor. CD 14 exists as both a cell surface receptor and soluble molecule found in normal blood serum, spinal fluid, etc. The level of sCD14 increase under some pathological conditions like infection, poly trauma, burns, and also SLE, etc. The increasing of sCD14 in serum is correlated to the development of some diseases. Now, sCD14 has been used as a marker for activation of monocyte/macrophage in monitoring the development of diseases. Less research on sCD14 has been done and no sCD14 ELISA Kit has been reported to be established up to now in China, while the Kit from US or Germany companies is too expensive to be widely used. For these reasons, we established a sandwich ELISA for the determination of soluble CD 14, which would be helpful to the research on sCD14 in China.A large amount of purified CD 14 protein was needed to prepare polyclonal and monoclonal antibodies which can recognize different epitopes of CD 14, and to study the relationship between CD 14 and Toll like receptors, and their roles in anti-infection immunity. Consequently, we purified native sCD14 from human plasma and expressed recombinant human CD 14 in Escherichia coli and Pichiapastoris. Our research can be divided into two parts: Part I Establishment of a sandwich ELISA for soluble CD14:Firstly, monoclonal and polyclonal antibodies to CD 14 were preparedand purified respectively. Ascitic fluid was purified with HiTrap protein G and 15mg purified MAb 60bca was obtained. Polyclonal antibody to CD 14 was got by immunizing rabbit with human monocyte transfected with human CD14 gene (THP1-CD14).Secondly, sandwich ELISA for the determination of soluble CD 14 was established using the antibodies described as above. The concentration of the capture mAb, polyclonal antibody and HRP-labeled goat-anti-rabbit IgG were determined. Serum from many healthy people was mixed together, which was used as the standard serum for ELISA system.Our kit has proven to be corresponding to that of foreign. The characteristic of the kit is high in specificity and sensitivity. The lowest detectable level is 5ng/ml, the range of detection is 5ng/ml~120ng/ml. The assay can be well repeated. The variation range is between 3.27%~9.72% and 6.7%~13.8% respectively in intra and inter assay. The level of sCD14 determined by our system is similar to that of IBL Kit.The serum level of sCD14 from 40 graduate students, 114 healthy old people, 100 patients of neuro-internal medicine and 300 patients with cardiovascular diseases were tested. The data shows that the concentration of serum sCD14 of normal people vary among different age groups. Compared with normal people, the level of serum sCD14 of patients with cerebral infarction or ACS increased significantly. Our research provides important evidence for further study in the mechanism of cerebral infarction and ACS. Part II Production of purified human CD14 proteinTo obtain native purified CD 14 protein, we separated sCD14 from human plasma using immune-affinity chromatography. The sCD14 affinity column was prepared by coupling purified mAb 60bca with CNBr-activated sepharose 4B, by which we got 400ug purified sCD14 from 200ml plasma.Although native sCD14 has more advantages on structure andbiological activities, the concentration is very low in human serum. So therecombinant CD 14 (rsCD14) had also been produced using expression systems of Escherichia coli and Pichiapastoris.Total RNA was isolated from THP1-CD14, and the gene of human CD 14 was obtained by RT-PCR. The sequence of PCR product is identical to that of CD 14 searched on GenBank. The products of the PCR were subcloned into pET28b and pPICZaA ve更多还原