發(fā)布時(shí)間：2018-04-22 19:42

  本文選題：S100A6 + 單克隆抗體��； 參考：《重慶醫(yī)科大學(xué)》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(gè)成員組成，其中21個(gè)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(diǎn)是穩(wěn)定性較差，故易發(fā)生染色體擴(kuò)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(xì)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(gè)細(xì)胞分裂增殖而形成單克隆細(xì)胞團(tuán)，可合成的針對(duì)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(guò)該技術(shù)可獲得既具有免疫的B淋巴細(xì)胞能產(chǎn)生抗體的能力，又具有骨髓瘤細(xì)胞不斷分裂能力的單克隆細(xì)胞。 單克隆抗體問(wèn)世后，由于其高特異性、高純度、高均一性的優(yōu)點(diǎn)已在醫(yī)學(xué)各個(gè)領(lǐng)域均有廣泛應(yīng)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(shí)，也是檢測(cè)疾病標(biāo)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(xué)療法中，充分利用單克隆抗體的導(dǎo)向作用，可將細(xì)胞信號(hào)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(yù)防、治療，特別是在腫瘤的免疫性診斷與治療中，開(kāi)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(dá)水平的升高，因此通過(guò)單克隆抗體的特異性對(duì)腫瘤中S100A6進(jìn)行快速檢測(cè)，有望建立起以免疫診斷技術(shù)為基礎(chǔ)的檢測(cè)方法，并進(jìn)一步實(shí)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(duì)其特異性進(jìn)行鑒定，為進(jìn)一步研發(fā)S100A6免疫學(xué)相關(guān)的檢測(cè)試劑和治療應(yīng)用奠定基礎(chǔ)。 方法： 1. GST-S100A6重組蛋白的原核表達(dá)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(dǎo)表達(dá)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(jì)測(cè)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(tái)過(guò)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(cè)小鼠血清抗體的效價(jià)，將SP2/0與血清效價(jià)高的脾細(xì)胞進(jìn)行融合，以GST-S100A6蛋白為包被原進(jìn)行正篩，再以GST為包被原反篩獲取雜交瘤細(xì)胞株。通過(guò)有限稀釋法進(jìn)行克隆化培養(yǎng)S100A6雜交瘤細(xì)胞株，并用不含標(biāo)簽的S100A6蛋白作為抗原通過(guò)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(shí)別,并在36KD處有陽(yáng)性條帶出現(xiàn)。 2.應(yīng)用上述原核表達(dá)所得GST-S100A6重組蛋白，對(duì)BALB/c小鼠進(jìn)行三次基礎(chǔ)免疫后，血清抗體效價(jià)檢測(cè)顯示在1：106以上，最后一次經(jīng)尾靜脈加強(qiáng)免疫，進(jìn)行細(xì)胞融合，最終篩選出21株雜交瘤細(xì)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(xì)胞株。Western blot法顯示其中1株單克隆細(xì)胞培養(yǎng)上清液內(nèi)可分泌出特異性抗體，即能識(shí)別S100A6蛋白。 結(jié)論： 本實(shí)驗(yàn)通過(guò)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(xì)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(hào)：1788632