S100A6蛋白单克隆抗体的制备CTGF对黑素色瘤细胞B16的迁移抑制作用及其与S100A6的关系

发布时间：2018-04-22 19:42

  本文选题：S100A6 + 单克隆抗体　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：S100蛋白是一类具有EF手型结构的钙结合蛋白家族，因其在中性饱和硫酸铵溶液中，溶解度为100%而得名。到目前为止，研究发现，，该家族至少由25个成员组成，其中21个成员的基因定位在人类染色体1q21上，此区染色体的显著特点是稳定性较差，故易发生染色体扩增和重排，因此该家族蛋白与肿瘤的发生发展有着密切关系。S100A6是该家族的成员之一，又称为钙周期蛋白(Calcyclin，Cacy)广泛分布于细胞内，最初由Kuznicki和Filipek在Ehrlich腹水瘤中发现。 单克隆抗体，由单个细胞分裂增殖而形成单克隆细胞团，可合成的针对一种抗原决定簇的抗体。1975年， Kohler和Milstein创建了杂交瘤技术，通过该技术可获得既具有免疫的B淋巴细胞能产生抗体的能力，又具有骨髓瘤细胞不断分裂能力的单克隆细胞。 单克隆抗体问世后，由于其高特异性、高纯度、高均一性的优点已在医学各个领域均有广泛应用。首先，单抗隆抗体是研究蛋白的功能、定位分布以及蛋白与蛋白之间的相互作用方面的一种重要的“工具”；同时，也是检测疾病标志物的重要工具，因此在疾病的诊断中发挥着十分重要的作用；在免疫治疗以及免疫-化学疗法中，充分利用单克隆抗体的导向作用，可将细胞信号通路封闭，或者将药物带至病灶部位，因此为人类疾病的诊断、预防、治疗，特别是在肿瘤的免疫性诊断与治疗中，开辟了新路径。 研究发现，在多数上皮源性肿瘤中出现S100A6表达水平的升高，因此通过单克隆抗体的特异性对肿瘤中S100A6进行快速检测，有望建立起以免疫诊断技术为基础的检测方法，并进一步实现肿瘤的靶向性治疗。 目的： 本课题拟制备S100A6钙结合蛋白的单克隆抗体，并对其特异性进行鉴定，为进一步研发S100A6免疫学相关的检测试剂和治疗应用奠定基础。 方法： 1. GST-S100A6重组蛋白的原核表达与鉴定： pGST-moluc和pGST-moluc-S100A6质粒转入BL21大肠杆菌并且用IPTG诱导表达后，冰上超声裂解，再用谷胱甘肽-琼脂糖4B球珠分离纯化GST蛋白和GST-S100A6蛋白，超微量分光光度计测定浓度后，经SDS-PAGE、Western-blot法鉴定重组蛋白，用0.22μm滤器在超净工作台过滤后分装，置于-80℃保存。 2．S100A6蛋白单克隆抗体制备、鉴定 用纯化的GST-S100A6重组蛋白免疫BALB/c小鼠，间接ELISA检测小鼠血清抗体的效价，将SP2/0与血清效价高的脾细胞进行融合，以GST-S100A6蛋白为包被原进行正筛，再以GST为包被原反筛获取杂交瘤细胞株。通过有限稀释法进行克隆化培养S100A6杂交瘤细胞株，并用不含标签的S100A6蛋白作为抗原通过Western blot法鉴定该抗体特异性。 结果： 1. Western blot结果显示，GST-S100A6重组蛋白可被商品化的S100A6抗体特异性地识别,并在36KD处有阳性条带出现。 2.应用上述原核表达所得GST-S100A6重组蛋白，对BALB/c小鼠进行三次基础免疫后，血清抗体效价检测显示在1：106以上，最后一次经尾静脉加强免疫，进行细胞融合，最终筛选出21株杂交瘤细胞株，用有限稀释法经克隆化培养后得到2株单克隆细胞株。Western blot法显示其中1株单克隆细胞培养上清液内可分泌出特异性抗体，即能识别S100A6蛋白。 结论： 本实验通过杂交瘤技术成功制备了1株能够分泌抗S100A6的单克隆抗体的杂交瘤细胞株。
[Abstract]:S100 protein is a family of calcium - binding proteins with EF - chiral structure . Its solubility is 100 % in neutral saturated ammonium sulfate solution . So far , it has been found that the family is composed of at least 25 members .

in 1975 , Kohler and Milstein created hybridoma technology , by which both that ability to produce antibodies to B - lymphocytes with immunity and the ability of myeloma cells to break apart are obtained .

The monoclonal antibody has been widely used in medical fields due to its high specificity , high purity and high homogeneity . First , the monoclonal antibody is an important " tool " for studying the function , localization and interaction of protein and protein .
At the same time , it is also an important tool to detect disease markers , so play a very important role in the diagnosis of diseases ;
In the immunotherapy and the immune - chemical therapy , the guiding action of the monoclonal antibody is fully utilized , the cell signal path can be blocked , or the medicine is brought to the lesion site , so that a new path is opened for the diagnosis , prevention and treatment of human diseases , in particular in the immunological diagnosis and treatment of tumors .

It is found that in most epithelial - derived tumors , the expression level is increased , so the specificity of the monoclonal antibody can be used for the rapid detection in the tumor , which is expected to establish the detection method based on the immunological diagnosis technique , and further realize the targeted treatment of the tumor .

Purpose :

In this study , the monoclonal antibody against the calcium binding protein of A6 A6 was prepared , and its specificity was identified . It lays a foundation for further research and development of the detection reagent and therapeutic application related to the immunology .

Method :

1 . The prokaryotic expression and identification of the recombinant protein GST - moluc and pGST - moluc were transformed into BL21 ( DE3 ) and induced by IPTG . The recombinant protein was purified by SDS - PAGE and Western - blot . After filtration , the recombinant protein was purified by SDS - PAGE and Western - blot , then the recombinant protein was separated and stored at -80 鈩

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