【作者】 师红磊；

【导师】 杨仕明；

【作者基本信息】 第三军医大学 ， 内科学， 2010， 硕士

【摘要】 研究背景和目的恶性肿瘤是当前严重影响人类健康的最重要的疾病之一。而肿瘤的侵袭和转移是导致肿瘤患者死亡的主要原因。肿瘤的转移过程非常复杂,目前认为转移性癌细胞侵袭转移主要通过以下三个步骤:1.肿瘤细胞通过细胞表面受体和细胞基质成分的粘附蛋白首先发生特异性结合,2.细胞外基质的水解酶降解过程,3.降解区发生瘤细胞的移动与侵袭。其中参与细胞外基质(ECM)降解过程中起作用的因素备受重视。肝素酶是1999年由四个不同的实验室克隆成功的一种肿瘤转移相关基因,肝素酶(heparanase, Hpa)是唯一能裂解ECM内蛋白多糖主要成分硫酸乙酰肝素蛋白多糖(heparan sulfate proteoglycans, HSPG)的内源性糖苷内切酶。研究表明肝素酶可以通过降解HSPG途径破坏ECM、BM的完整性以及促进肿瘤新生血管的生成而促进肿瘤的转移。越来越多的研究证实,肝素酶在几乎所有的中晚期恶性肿瘤中都高表达,与肿瘤患者的不良预后密切相关,抑制肝素酶的表达可以明显减少肿瘤的侵袭和转移。因此,肝素酶有可能作为肿瘤诊断与治疗的新靶点。单克隆抗体(Monoclonal Antibody,McAb)是单个B淋巴细胞克隆所分泌的抗体。抗体主要由B淋巴细胞合成,每个B淋巴细胞有合成一种抗体的遗传基因。当机体受抗原刺激时,抗原分子上的许多决定簇分别激活各个具有不同基因的B细胞。被激活的B细胞分裂增殖形成该细胞的子孙,即克隆。如果选出一个合成一种抗体的细胞进行培养,就可得到由单细胞经分裂增殖而形成细胞群,即单克隆。单克隆细胞所合成的抗体即为单克隆抗体。1975年Kohler和Milstein首先报道,用细胞杂交技术,使经绵羊红细胞(SRBC)免疫的小鼠脾细胞,与小鼠骨髓瘤细胞融合,创建了第一个B细胞杂交瘤细胞株,获得了抗SRBC的单克隆抗体。这是免疫学,乃至医学史上的一个里程碑。单克隆抗体的特点是:理化性状高度均一、生物活性单一、与抗原结合的特异性强、便于人为处理和质量控制,并且来源容易。这些优点使它一问世就受到高度重视,并成为解决生物学和医学等许多重大问题的重要手段。单克隆抗体具有高度特异性、高纯度特点,使其在很大浓度范围内获得低背景,所以在组织免疫染色中特别有用,还可以应用于免疫沉淀,免疫印迹技术,并且都具有特异性高,背景低的优点。另外单抗还可以用来进行免疫亲和纯化抗原;对单克隆抗体进行标记还可以用做快速诊断以及疫苗的研究等。目前有关肝素酶的单克隆抗体多见于国外公司生产,我国尚无商品化的肝素酶单克隆抗体。单纯的从国外进口该抗体,价格昂贵,成本高。基于以上认识,本研究拟构建人肝素酶高效表达基因,以获得人肝素酶重组蛋白,再利用获得的高纯度肝素酶蛋白作为免疫原,采用常规免疫程序和杂交瘤技术筛选人肝素酶特异性单克隆抗体,并利用免疫组化、ELISA及Western Blot等技术对获得的人肝素酶单克隆抗体进行鉴定,最后获得人肝素酶特异性的单克隆抗体,为以后肝素酶单克隆抗体在临床诊断以及抗体介导的肿瘤靶向治疗提供实验依据。方法1.将人肝素酶全长cDNA克隆到pET30a质粒,进一步转化入宿主菌BL21 (DE3)中进行原核表达,采用Ni2+柱纯化对人肝素酶重组蛋白进行纯化,对表达的人肝素酶重组蛋白进行质谱分析;2.将获得的肝素酶蛋白作为免疫源免疫Balb/c小鼠获得经免疫后能产生肝素酶抗体的B淋巴细胞;采用杂交瘤技术制备分泌肝素酶抗体的杂交瘤细胞株;采用有限稀释法筛选分泌抗体呈强阳性的克隆,并对上清液进行ELISA检测,鉴定筛选出的阳性克隆;3.采用HIS亲和层析以及复性纯化技术纯化肝素酶单克隆抗体;获得的抗体采用Western Blot技术、免疫组化技术及ELISA技术检测制备的肝素酶单克隆抗体的特异性和抗体的效价。结果1.我们采用PCR技术克隆了人肝素酶蛋白编码基因。经测序与GENBANK提供的基因序列完全一致。将克隆的人肝素酶基因序列克隆入原核表达质粒pET30a构建人肝素酶原核表达重组质粒;经1.0mmol/L IPTG 37℃诱导表达5h,pET30a-Hpa在BL21中表达融合蛋白,分子量为63KD左右,表达量占菌体总蛋白的30 %以上。经鉴定表达蛋白破菌后主要在沉淀中,为包涵体表达。采用HIS亲和层析及复性纯化方法,纯化获得人肝素酶重组蛋白;2.利用纯化的人肝素酶蛋白免疫原采用常规免疫程序和杂交瘤技术,经细胞融合,筛选出15株抗人肝素酶的特异性单克隆抗体,经亚类鉴定其中有3株为适合应用于诊断试剂的IgG1;3.经免疫组化、ELISA、及Western等多种手段对获得的肝素酶单克隆抗体进行特异性分析和效价鉴定,结果显示所制备的3株单抗为人肝素酶特异的,且效价较高的单克隆抗体。结论1.我们采用PCR技术克隆了人肝素酶蛋白编码基因。获得了高纯度的人肝素酶蛋白,获得的肝素酶蛋白经鉴定其测定分子量与理论分子量相符,可以用于肝素酶单克隆抗体的制备;2.成功制备了3株人肝素酶特异性的单克隆抗体,获得的三株人肝素酶单克隆抗体经ELISA、Western Blot和免疫组化等技术检测,证实其特异性高,并具有较高的效价,该研究结果提示我们制备的肝素酶单克隆抗体可能用于以肝素酶为靶点的肿瘤的诊断和治疗。更多还原

【Abstract】 Background and ObjectiveCancer, one of the most important diseases, has serious impacts on human health at present. The tumor invasion and metastasis is the main reason leading to the death of cancer patients. Metastasis process is very complex, now that the metastatic cancer cell invasion and metastasis, mainly through the following three steps: 1. tumor cell through cell surface receptors and cell adhesion to extracellular matrix protein first with specificity, 2. extracellular matrix hydrolase degradation, 3. degradation occurred tumor cell migration and invasion. And the factors involved in extracellular matrix (ECM) in the degradation process have attracted increasing attention.Heparanase in 1999 by four different laboratories successfully cloned a tumor metastasis-related genes, heparanase (Hpa) is the only way to cracking the major ECM components within the proteoglycan heparan sulfate proteoglycan (HSPG) of endogenous glucosidase enzyme. Studies show that the degradation of HSPG by heparanase way undermines ECM, BM integrity and the promotion of tumor angiogenesis and metastasis. More and more studies confirm that in almost all of heparanase in the expression of both advanced cancer and poor prognosis of cancer patients, and inhibition of heparanase expression can significantly reduce tumor invasion and metastasis. Thus, heparanase may serve as cancer diagnosis and therapeutic target.Monoclonal antibody (Monoclonal Antibody, McAb) is a single B lymphocyte clone secreting antibodies. Each B lymphocyte has synthesized a kind of antibody genes. When the body is stimulated by the antigen, the antigen molecules were many determinants each with different genes activated B cells. Activated B cell was proliferation of the cells in the formation of children, which is called cloning. If the election of a synthesis of an antibody to the cell culture, can be obtained from the single cell formed by the proliferation of the cell groups, which is called monoclonal. In 1975, Kohler and Milstein first reported that by the sheep red blood cells (SRBC) immunization of mouse spleen cells with mouse myeloma cell fusion, creating the first B cell hybridoma cell lines obtained anti-SRBC monoclonal antibodies. This is immunology, and even a milestone in the history of medicine. Monoclonal antibody is characterized by: a high degree of homogeneous physical and chemical properties, biological activity of single, antigen binding specificity, and easy handling and quality control people. These advantages make it come out to be a high priority, and the solution to biology and medicine, an important means many important issues. Monoclonal antibodies are highly specific, high purity characteristics, so that within a large concentration of low background, is particularly useful in immunohistochemistry, immunoprecipitation and immunoblotting, and have high specificity, the advantages of low background. Another monoclonal antibody can also be used for immunoaffinity purified antigen; the monoclonal antibody markers can also be used for rapid diagnosis and vaccine research. At present, the heparanase monoclonal antibodies prevalent in the foreign company, there is no commercialization of our heparanase monoclonal antibodies. The monoclonal antibodies imported from abroad are expensive and high cost.Based on the above points, this study was to build effective human heparanase gene expression, to obtain recombinant human heparanase, and then use access to high-purity protein as immunogen heparanase, routine immunization procedures and the hybridization technique to screen a human heparanase-specific monoclonal antibody. Immunohisto- chemistry, ELISA and Western Blot techniques were used on the available human heparanase monoclonal antibodies, and finally get specific human heparanase monoclonal antibody, for the future of heparanase monoclonal antibody in clinical diagnosis and antibody-mediated tumor targeted therapy to provide experimental basis.Methods1. The human heparanase full cDNA was cloned into pET30a plasmid, and further transformed into host strain BL21 (DE3) in prokaryotic expression. The human heparanase recombinant protein was purified by Ni2+ column purification, and expression of human heparanase recombinant protein was analyzed by mass spectrometry.2. Heparanase protein was used to immune Balb / c mice, producing antibodies of the B lymphocyte heparanase. Hybridoma cell lines secreting heparanase antibody were prepared; by limiting dilution screening of antibodies was strongly positive clone, and supernatant ELISA, positive identification of selected clones.3. HIS affinity chromatography purification and refolding purification was used to purify heparanase monoclonal antibody. Antibody obtained was used Western Blot technique, immunohistochemistry and ELISA to detect heparanase preparation of monoclonal antibody specificity and the efficiency of antibody price.Results1. We used PCR cloning of human heparanase protein coding genes. The sequence of the gene sequence with GENBANK provides exactly the same. The cloning of human heparanase gene was cloned into the prokaryotic expression vector pET30a heparin was constructed recombinant prokaryotic expression plasmid; by 1.0mmol / L IPTG 37℃induced 5h, pET30a-Hpa expression of fusion protein in BL21, a molecular weight of about 63KD, total bacterial protein expression more than 30%. Protein was identified after breaking the main bacteria in the deposition, expressed as inclusion bodies. Purified recombinant human heparanase protein was obtained by HIS affinity chromatography and refolding purification.2. Purified human heparanase protein immunogen was using conventional immunization procedures and hybridoma techniques, by cell fusion, screening out 15 specific anti-human heparanase monoclonal antibodies, identified by the sub-class of which 3 strains suitable for use in the diagnosis reagent IgG1.3. By immunohistochemistry, ELISA, and Western to get the heparin on the enzyme specificity of monoclonal antibodies and titers of identification, the results show that the prepared 3 heparanase specific antibody, and the high titer monoclonal antibodies.Conclusion1. We used PCR cloning of human heparanase protein coding genes. High-purity human heparanase protein was obtained by the identification of the heparanase protein molecular weight determination consistent with the theoretical molecular weight, which can be used for heparanase monoclonal antibodies.2. We have successfully prepared three specific human heparanase monoclonal antibodies. These three human heparanase monoclonal antibodies were identified by ELISA, Western Blot and immunohistochemistry, confirming the high specificity and high titer. The study suggests that our preparation of heparanase monoclonal antibodies may be used to heparanase as a target for cancer diagnosis and treatment.更多还原