本文選題：S100A6 + 單克隆抗體�。� 參考：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：S100蛋白是一類具有EF手型結(jié)構(gòu)的鈣結(jié)合蛋白家族，因其在中性飽和硫酸銨溶液中，溶解度為100%而得名。到目前為止，研究發(fā)現(xiàn)，，該家族至少由25個成員組成，其中21個成員的基因定位在人類染色體1q21上，此區(qū)染色體的顯著特點是穩(wěn)定性較差，故易發(fā)生染色體擴增和重排，因此該家族蛋白與腫瘤的發(fā)生發(fā)展有著密切關(guān)系。S100A6是該家族的成員之一，又稱為鈣周期蛋白(Calcyclin，Cacy)廣泛分布于細胞內(nèi)，最初由Kuznicki和Filipek在Ehrlich腹水瘤中發(fā)現(xiàn)。 單克隆抗體，由單個細胞分裂增殖而形成單克隆細胞團，可合成的針對一種抗原決定簇的抗體。1975年， Kohler和Milstein創(chuàng)建了雜交瘤技術(shù)，通過該技術(shù)可獲得既具有免疫的B淋巴細胞能產(chǎn)生抗體的能力，又具有骨髓瘤細胞不斷分裂能力的單克隆細胞。 單克隆抗體問世后，由于其高特異性、高純度、高均一性的優(yōu)點已在醫(yī)學各個領(lǐng)域均有廣泛應用。首先，單抗隆抗體是研究蛋白的功能、定位分布以及蛋白與蛋白之間的相互作用方面的一種重要的“工具”；同時，也是檢測疾病標志物的重要工具，因此在疾病的診斷中發(fā)揮著十分重要的作用；在免疫治療以及免疫-化學療法中，充分利用單克隆抗體的導向作用，可將細胞信號通路封閉，或者將藥物帶至病灶部位，因此為人類疾病的診斷、預防、治療，特別是在腫瘤的免疫性診斷與治療中，開辟了新路徑。 研究發(fā)現(xiàn)，在多數(shù)上皮源性腫瘤中出現(xiàn)S100A6表達水平的升高，因此通過單克隆抗體的特異性對腫瘤中S100A6進行快速檢測，有望建立起以免疫診斷技術(shù)為基礎(chǔ)的檢測方法，并進一步實現(xiàn)腫瘤的靶向性治療。 目的： 本課題擬制備S100A6鈣結(jié)合蛋白的單克隆抗體，并對其特異性進行鑒定，為進一步研發(fā)S100A6免疫學相關(guān)的檢測試劑和治療應用奠定基礎(chǔ)。 方法： 1. GST-S100A6重組蛋白的原核表達與鑒定： pGST-moluc和pGST-moluc-S100A6質(zhì)粒轉(zhuǎn)入BL21大腸桿菌并且用IPTG誘導表達后，冰上超聲裂解，再用谷胱甘肽-瓊脂糖4B球珠分離純化GST蛋白和GST-S100A6蛋白，超微量分光光度計測定濃度后，經(jīng)SDS-PAGE、Western-blot法鑒定重組蛋白，用0.22μm濾器在超凈工作臺過濾后分裝，置于-80℃保存。 2．S100A6蛋白單克隆抗體制備、鑒定 用純化的GST-S100A6重組蛋白免疫BALB/c小鼠，間接ELISA檢測小鼠血清抗體的效價，將SP2/0與血清效價高的脾細胞進行融合，以GST-S100A6蛋白為包被原進行正篩，再以GST為包被原反篩獲取雜交瘤細胞株。通過有限稀釋法進行克隆化培養(yǎng)S100A6雜交瘤細胞株，并用不含標簽的S100A6蛋白作為抗原通過Western blot法鑒定該抗體特異性。 結(jié)果： 1. Western blot結(jié)果顯示，GST-S100A6重組蛋白可被商品化的S100A6抗體特異性地識別,并在36KD處有陽性條帶出現(xiàn)。 2.應用上述原核表達所得GST-S100A6重組蛋白，對BALB/c小鼠進行三次基礎(chǔ)免疫后，血清抗體效價檢測顯示在1：106以上，最后一次經(jīng)尾靜脈加強免疫，進行細胞融合，最終篩選出21株雜交瘤細胞株，用有限稀釋法經(jīng)克隆化培養(yǎng)后得到2株單克隆細胞株。Western blot法顯示其中1株單克隆細胞培養(yǎng)上清液內(nèi)可分泌出特異性抗體，即能識別S100A6蛋白。 結(jié)論： 本實驗通過雜交瘤技術(shù)成功制備了1株能夠分泌抗S100A6的單克隆抗體的雜交瘤細胞株。
[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

本文編號：1788632