【作者】 许鹏；

【导师】 窦科峰； 陈志南；

【作者基本信息】 第四军医大学 ， 外科学， 2002， 硕士

【摘要】 目的：单克隆抗体HAb18以其对肝癌细胞的高亲和力、高特异性，在肝癌的诊断、治疗中显示出巨大的优势和潜力，是我国唯一被批准进入二期临床试验的单抗制剂。但是，HAb18单抗本身并无治疗功能。目前核素交联的HAb18单抗在临床上难以被患者和医务工作者广泛接受。因此，制备新型具有高度特异性而又不须交联核素即裸抗体就有治疗功能的HAb18单抗成为HAb18研究的一个新方向和切入点。构建CD147/HAb18G基因原核表达载体并尝试在原核表达系统表达CD147/HAb18G蛋白，大量制备供筛选新型单抗的CD147/HAb18G抗原是本课题的首要目的。同时，原核表达的CD147/HAb18G蛋白是否具有抗原性，是决定利用原核表达的HAb18G蛋白筛选新型HAb18单抗能否取得突破性进展的关键所在。因此，用纯化的CD147/HAb18G原核表达蛋白进行抗原性检测，是本课题的核心内容。天然CD147/HAb18G蛋白具有刺激肝癌及其周围组织中成纤维细胞(fb)产生基质金属蛋白酶MMPs(Matrix Metalloproteinases)的作用。MMPs具有破坏局部组织结构和基底膜屏障、促进肿瘤血管生成的作用。但 第四军医大学硕士学位论文 CD147／HAbl8G分子诱导MMPS产生的机制及是否具有其它功能目前尚 不清楚。因此，对原核表达的纯化的CD147／HAbl8G蛋白生物活性进行 初步检测，为进一步探讨该蛋白在肝癌转移中的作用及其他生物学功能 的研究奠定基础是本课题的另一目的。 方法：本实验共分四个步骤完成 （一）CD147／HAblSG基因原核表达载体的构建 用PCR 方法扩增HAb ISG全长CDNA，将片段CDNA 及质粒载体 pRSET｛双酶切后用T；DNA连接酶将二者连接，构建表达载体的。将表 达载体进行酶切鉴定，并通过测序来验证插入片段的正确性。 （H）CD 47AfAb SG基因在大肠杆菌BLZ VDE3）中的高效表达 将重组表达载体 pRSET－C一HAbl8G转染 E．COlt BLZI（DE3） IPTG 诱导下表达蛋白，SDS－PAGE检测蛋白表达，激光薄层扫描检测蛋白表 达量占菌体蛋白总量的百分比。 （三）CD 47MAb SG融合蛋白的纯化及鉴定 将表达的融合蛋白用Ni－NTA柱亲和层析纯化，SDS－PAGE检测纯 化效果，观察纯化后融合蛋白的分子量验证表达蛋白的正确性。 （四）CD 47fHAb SG原核表达蛋白的生物学活性检测 1．利用纯化后融合蛋白进行酶联免疫吸附测定。将 CD147MAbl8G蛋白包被于ELISA板；以HAbls单抗为一抗 倍比稀释，以 E－羊抗鼠 IgG为酶标H抗，于 590 urn读取光吸 收（A）值。 2．利用纯化后融合蛋白进行明胶酶谱测定。 将纯化的CD 47／HAb18G原核表达蛋白加入成纤维细胞（fbX 设立对照组，培养细胞后收集上清，梯度离心除去细胞碎片， 4 第四军医大学硕士学位论文 沉淀蛋白后离心收集沉淀，透析除盐。上样于含0l％明胶的 6％聚丙烯酞胺凝胶进行SDS－PAGE。电泳结束后依次洗脱、孵 育，考马斯亮蓝染色、脱色，观察结果。 结果： 1．HAb SG基因真核表达载体的构建 l．l将PCR扩增的CD147／HAbl8G片段双酶切后，电泳出现一条清 晰的 17kb大小 DNA条带，为插入的 CD147／HAbl8G CDNA序 列；pRSET（经双酶切后，电泳出现一条带，分子量约3刀kb。 1．2将重组的表达载体双酶切鉴定，结果表明：CD147／HAbl8G CDNA片段正确插入载体中的酶切位点，大小为1．7kb。 1．3测序结果表明，插入序列与己知序列结果完全相符，证明载体构 建是成功的。 2．CD147MAbl8G基因在大肠杆菌BLZI①E3）中的高效表达，融合蛋白 的纯化及鉴定 2刁用 pRSET－C—HAbl8G转化感受态E．COlt BLZI（DE3X 经 IPTG诱 导表达后，以 12％SDS－PAGE捡测。与对照相比，在相对分子 量（Mr）为34．6KD处出现一条深染新生条带，与预期的甸 HAbl8G融合蛋白大小相符，表明CD147／HAbl8G融合蛋白原核 表达是成功的。 2二激光薄层扫描分析显示：CD 47／HAbl8G的表达量占菌体蛋白总更多还原

【Abstract】 AIM: Monoclonal antibody HAblS is a high affinity and specific anti-hepatoma antibody. It has a great advantage and potential in human hepatoma carcinoma diagnosis and therapy. At present, it is the only one mAb that being approved into phase II clinical test in China. But mAb HAblS itself has no therapeutic function and the radioisotope linked HAblS is not broadly accepted by patients and physicians. So developing high specific therapeutic antibodies having the same target as HAblS is a new research direction. Construct HAbISO/CD 147 prokaryotic expression vector and try to get enough HAbl8G/CD147 protein so that can be used into developing antibodies is the primary aim of this project. Whether HAbl8G/CD147 protein expressed by prokaryotic expression system has immunogenicity or not is the key question, because only the expressed protein with immunogenicity that can be used screening new antibodies. So detecting the immunogenicity of HAbl8G/CD147 expressed by prokaryotic expression system is the second aim of the project. Finally, the natural HAbl8G/CD147 protein play an important role in infiltration and metastasisof hepatoma cells, as it can induce fibroblast cells around the hepatoma tissue secreting MMPs (Matrix metalloproteinases). MMPs have the ability of destroying local histological construct and basement membrane barrier. However, the mechanism of HAbl8G/CD147 inducing MMPs and other functions are not clear. So doing the functional experiments about the purified HAbl8G/CD147 expressed by prokaryotic expression system will help us understand the role of it in metastasis and lay the foundation of further research of this protein. Methods: The experiments includes four parts1. Construction of prokaryotic expression vector of HAbl 8G/CD147gene. The full length cDNA of HAbl8G/CD147 was obtained by PCR method, recombinant vector pBluescript/HAblSG was the template. The enzyme site BamH I and Xho I were designed in primers. The fragment of the cDNA cut by BamH I and Xho I was linked with the prokaryotic expression vector pRSET-C cut with the same enzymes. The constructed vector was identified by restriction endonucleases digesting and sequencing.2. High efficient expression of HAbl8G/CD147 gene in E.coli BL21(DE3), purification and identification of fusion protein.The E.coli BL21(DE3) was transformed by the recombinant prokaryotic expression vector pRSET-C ?HAblSG. The protein was induced by IPTG (0.5m mol/1), the control were the expression induced without IPTG and the E.coliBL21(DE3) transformed by the empty vectorpRSET-C. The expression products were identified by SDS-PAGE and Laser Thin Layer Scan. 3. Purification and identification of HAb 18G/CD147 fusion protein.The expression products were purified by Ni-NTA affmity-chromatography. The purified protein was further identified by SDS-PAGE.4 . Functional experiments of HAbl8G/CD147 prokaryotic expression protein in vitro.4.1 ELISA assay of purified fusion proteinDilute the protein from 1:250 concentration in 96 well plate as the antigen. Add HAb 18 as the first antibody and enzyme linked goat-anti-mouse antibody as the second antibody. Colorize with OPD, read data by ELISA assay machine.4.2 Gelatin Zymography of the purified fusion proteinFive groups of cells were designed :(l)fb;(2) HHCC; (3) HHCC+fb (4)purified fusion pratein + fb; (5)positive control group (purified eukaryotic expression protein +fb). Collect the culture supernatant, abandon cells debris by gradient centrifugation, collect sedimentation after deposition and desalt. 10 u 1 samples individually was add to SDS-PAGE electrophoresis (the glue contained 0.1% gelatin). After electrophoresis, elute, incubate, stain discolor in turn.Results:1. Construction of prokaryotic expression vector of HAbl8G/CD147.1.1 Electrophoresis showed a 1.7kb band by cutting PCR products and a 3.0kb band by cutting vector pRSET-C.1.2 Electrophoresis showed a 1.7kb band and a 3.0kb band by cutting vector pRSET-C ?HAblSG wit更多还原