【作者】 别彩群；

【导师】 杨冬华；

【作者基本信息】 暨南大学 ， 内科学， 2009， 博士

【摘要】 研究背景和意义:肝细胞癌(Hepatocellular carcinoma,HCC)是全球第五常见的癌症,是我国癌症中的第二号杀手,全球50%以上的肝癌发生在我国。目前传统的治疗如外科手术及放化疗等没有理想的疗效。单链抗体(Single chain variablefragment,scFv)分子量小、穿透力强、体内半衰期短、免疫原性低并且具有亲本抗体相同的抗原结合能力,可能成为肝癌靶向治疗的理想载体。我们课题组采用噬菌体抗体展示技术成功筛选出1株特异性抗肝癌噬菌体scFv 4-16(GenBank:DQ640759),并对其进行了体外亲和力成熟及人源化改造,获得了具有较高亲和力和较低免疫原性的scFv。初步研究显示特异性抗肝癌scFv 4-16对肝癌细胞及肝癌组织抗原具有较好的特异性,拥有较好的研究开发和临床应用潜力。但scFv是单价抗体,仍存在着亲和力较弱、稳定性较差、体内清除过快、在大肠杆菌中分泌表达量过小等不足。因此,本研究重点构建亲和力高和稳定性好的抗肝癌单链抗体二聚体,并实现其在毕赤酵母中的高效表达,制备单链抗体二聚体纳米颗粒,观察其对体外肝癌细胞增殖的影响。为探讨单链抗体对肝癌的放射免疫显像诊断和靶向治疗价值提供依据。第一章抗肝癌噬菌体单链抗体二聚体的构建、表达与鉴定目的:构建抗肝癌噬菌体单链抗体二聚体,并在大肠杆菌中可溶性表达,鉴定其生物活性。方法:将我室构建的抗肝癌噬菌体scFvDM连接肽的长度从15个氨基酸残基缩短至3-5个,克隆至载体pCANTAB5E,转化感受态大肠杆菌TG1并测序,对测序正确的阳性克隆予辅助噬菌体感染,制备噬菌体抗体,检测其相对亲和力,将亲和力指数最高的噬菌体抗体在大肠杆菌HB2151中进行诱导可溶性表达,采用SDS-PAGE、Western blotting鉴定表达产物,并进行浓度及特异性检测。结果:测序结果显示成功构建三株抗肝癌噬菌体单链抗体二聚体(scFv-3、scFv-4、scFv-5),其亲和力指数是亲本抗体的3.5-6倍。将亲和力指数最高的一株二聚体(scFv-5,命名为BDM3)在大肠杆菌HB2151中可溶性表达,通过SDS-PAGE和Western blotting检测到目标蛋白带;ELISA结果显示:表达产物具有与肝癌细胞特异性结合活性,与胎肝细胞、胃癌细胞等均无交叉反应,但抗体表达的量较低,浓度仅为100μg/L菌液,不能满足后续的研究需要。结论:成功构建了抗肝癌噬菌体单链抗体二聚体基因,并获得可与肝癌细胞特异结合的高亲和力的抗肝癌单链抗体二聚体,为后续实现抗体在酵母中大量表达奠定基础。第二章抗肝癌单链抗体二聚体在酵母中表达及其生物活性的鉴定目的:实现抗肝癌单链抗体二聚体在毕赤酵母中高效表达,并鉴定所表达抗体的生物活性。方法:构建酵母表达载体pGAPZαA-BDM3,转化感受态大肠杆菌DH5α,选择测序正确的阳性克隆进行扩增后,电转化酵母细胞,表达抗体二聚体,对表达抗体进行纯化、二聚体分子量及形成比例、浓度检测,并鉴定其对肝癌细胞的结合活性和稳定性,免疫组化检测二聚体对肝癌组织抗原的特异性。结果:测序显示成功构建酵母表达载体pGAPZαA-BDM3,表达96小时抗体收获量最大,纯化后的抗体经凝胶过滤色谱检测,二聚体的分子量为62kD,二聚体形成比例为92%。二聚体表达量为30mg/L菌液,为大肠杆菌的300倍。抗肝癌单链抗体二聚体BDM3与三种肝癌细胞结合,而与正常肝细胞不结合,结合效价为1:128。稳定性实验证实抗肝癌单链抗体二聚体BDM3在37℃孵育16小时后与肝癌细胞的结合活性下降到50%,孵育48小时后完全失去结合活性,具有很好的稳定性。免疫组化显示二聚体与肝癌组织结合的阳性率比肝硬化、胃癌、肠癌、正常肝组织高,差异有统计学意义。结论:成功制备了高表达量、高特异性、较好的活性及稳定性的抗肝癌单链抗体二聚体BDM3,为制备免疫纳米颗粒及开展肝癌的放射免疫诊断和靶向治疗奠定了基础。第三章免疫纳米颗粒制备、检测及其对肿瘤细胞增殖的影响目的:制备高分子免疫纳米颗粒,并观察其对肝癌细胞增殖的影响。方法:采用离子交联的方法,以壳聚糖水溶性衍生物多糖为基材,将已获得的抗肝癌单链抗体二聚体制备成免疫纳米颗粒,检测纳米颗粒的表征、包封率及载药量,并通过MTT法观察免疫纳米颗粒对肝癌细胞株增殖的影响。结果:制备粒径为100-200nm的抗肝癌单链抗体二聚体高分子纳米颗粒,最佳包封率为53%,载药量为每毫克75微克抗体二聚体,高分子纳米颗粒对肿瘤细胞无毒性,但抗肝癌单链抗体二聚体高分子纳米颗粒显示较好的抗肿瘤作用,其对肝癌细胞的抑制率为34%左右,且有浓度依赖性。结论:成功制备了抗肝癌单链抗体二聚体高分子纳米颗粒,初步应用具有抑瘤性,为下一步开展体内肝癌的放射免疫诊断和靶向治疗奠定了基础。更多还原

【Abstract】 Background Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death in our country.Traditional therapies,such as resection,chemotherapy and radiotherapy have not satisfactional efficacy.Single chain variable fragments(scFv) can be a good vector of targeted therapy for HCC as a result of their small molecule weight,strong tumor tissue penetration,weak immunogenicity and short half-lives in blood,as well as possessing the same binding ability and specificity as the whole antibody.My work team had obtained a scFv against hepatocellular carcinoma(scFv 4-16,GenBank:DQ640759) through the phage display antibody library technology.After reconstruction of affinity maturation and humanization of scFv,we have attained a scFv with high affinity to HCC and low immunogenicity.However,scFv is monovalent antibody with the shortcoming of low affinity,bad stability,too rapid clearance from the circulation and small-scale preparation in bacterial expression system.This study is to construct the non- covalent single-chain Fv dimers for Hepatocellular carcinoma,express the dimmers in Yeast pichlia pastores,prepare the immunonanoparticles and investigate their effects on HCC cells.It may provide an evidence for hepatocellular carcinoma targeted diagosis and therapy in vovo in the future.Chapter 1 Construction,expression and assay of single-chain Fv dimers for hepatocellular carcinoma in E.coll.Objective:To construct single-chain Fv dimers for hepatocellular carcinoma,express in E.coli and assay the biological activities.Methods:We had reported the construction, screening and humanization of the single-chain Fv for HCC.The linker between VH and VL genes were shortened to 3～5 amino acid residues,cloned into the vector of pCANTABSE.The recombinant vector plasmids were transformed into TG1 cells and sequenced.The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibody.ELISA was used to analyze the binding activity of phage to HCCs.HB2151 were infected with the phage antibodies which had the best binding affinity to hepatocellular carcinoma to express single-chain Fv dimers. Expressed products were identified by SDS-PAGE,West-blotting and ELISA.Results: Three single-chain Fv dimers genes(scFv-3,scFv-4,scFv-5) had been constructed successfully with the binding ability 3.5-6 folds to hepatocellular carcinoma comparing with their parental scFv.The single-chain Fv dimmer(scFv-5,termed BDM3) with the best binding ability was expressed.There was a strip considered to be aim protein in the results of SDS-PAGE and Western blotting.The concentration of BDM3 was only 100μg /L,which was not enough for the following research.Conclusions:The single-chain Fv dimers had been constructed and expressed successfully in E.coli.The expressed products have better antigen binding activity.That was a basic work for the following experiments.Chapter 2 Expression and assay of single-chain Fv dimers for hepatocellular carcinoma in Yeast pichlia pastores.Objective:To express dimers BDM3 in Yeast pichia pastores and assay its biological activities.Methods:We had constructed pGAPZαA-BDM3 and transformed it into DH5α.The recombinant vector plasmids were sequenced and transformed to Yeast pichlia pastores GS115.BDM3 was expressed in Yeast pichlia pastores.Expressed products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography(SEC),ELISA and immunohistochemistry.Results:SDS-PAGE and Western blotting had shown that the dimers BDM3 was successfully expressed in Yeast pichlia pastores.The SEC results suggested the molecular weight of the expressed products were about 62 kD with 92%dimerization.The concentration of BDM3 was 30mg/L,which was 300 times as many as the products in E.coli.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv,even after 16 h incubation at 37℃still having 50%binding activity.The purified dimmers were specially binding to tumor antigen of HCC.Conclusions:The functional scFv dimers against hepatocellular carcinoma was achieved,and it was essential to make use of the dimers in hepatocellular carcinoma targeted diagosis and therapy. Chapter 3 Preparation and assay of immunonanoparticles and investigation of their effects on HCC cellsObjective:To obtain and identify the polyelectrolyte nanoparticles,and investigate the effect of BDM3-loading nanoparticles on HCC cells.Methods:By means of IR and TEM,the physicochemical characteristics of the polyelectrolyte nanoparticles were manifested,including the the particle size,morphology and the concentration of loaded protein on the particle size,protein entrapment efficiency of the nanoparticles. Furthermore,we investigated the effect of BDM3-loading nanoparticles on HCC cells. Results:The particle size is ranged from 100nm to 200nm and has a suitable concentration of loaded protein on the particle size with a 53%entrapment efficiency of BDM3.There was a significant inhibition of BDM3-loading nanoparticles on HCC cells growth.Conclusions:A targeted BDM3 nanopaticle has been obtained and it is a basic work for HCC targeted diagosis and therapy in vivo in the future research.更多还原