本文選題：c-myc　切入點(diǎn)：免疫　出處：《遼寧師范大學(xué)》2011年碩士論文

【摘要】：原癌基因c-myc是myc家族的重要成員之一,是髓細(xì)胞性白血病病毒的癌基因v-myc的細(xì)胞同類物,定位于染色體8q24.12-8q24.13,其編碼產(chǎn)物c-myc蛋白的分子量為62kDa。c-myc基因的功能甚多,它既是一種可易位基因,又是一種受多種物質(zhì)調(diào)節(jié)、可使細(xì)胞無限增殖、獲永生化功能、促進(jìn)細(xì)胞分裂的可調(diào)節(jié)基因,在很多腫瘤中都高表達(dá)。在正常細(xì)胞中的c-myc原癌基因一旦被激活,轉(zhuǎn)為癌基因,活化轉(zhuǎn)錄和翻譯,c-myc mRNA和c-myc蛋白的表達(dá)便異常增高,使細(xì)胞脫離正常生長的調(diào)節(jié)限制而具有高度增生潛能,向惡性表型轉(zhuǎn)化,抑制細(xì)胞分化,促進(jìn)細(xì)胞惡變,最后導(dǎo)致腫瘤的發(fā)生。鑒于c-myc的過表達(dá)與腫瘤發(fā)生呈高度正相關(guān),c-myc抗血清可以應(yīng)用于腫瘤的診斷,本研究通過制備c-myc單克隆抗體,為腫瘤診斷試劑盒的研發(fā)提供基礎(chǔ)實(shí)驗(yàn)數(shù)據(jù)。用原核表達(dá)及純化的c-myc蛋白抗原免疫3只健康雌性BALB/c小鼠,3次免疫后取小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞以10:1比例進(jìn)行細(xì)胞融合,再經(jīng)HAT選擇培養(yǎng)基篩選、間接ELISA方法檢測、有限稀釋法克隆,最終獲得2株能夠穩(wěn)定分泌抗c-myc特異性單克隆抗體的雜交瘤細(xì)胞株,并分別命名為SHH-1H11和SHH-2A9。抗體亞型鑒定表明,SHH-1H11、SHH-2A9分別為IgG1亞類和IgM。經(jīng)BCA Protein Assay Reagent Kit檢測顯示,雜交瘤細(xì)胞SHH-1H11上清和腹水的抗體濃度分別為0.47mg/ml和2.51mg/ml,SHH-2A9的上清和腹水的抗體濃度分別是0.49mg/ml和2.45mg/ml。ELISA檢測結(jié)果顯示,這兩株雜交瘤細(xì)胞培養(yǎng)上清效價(jià)分別為1:10240、1:5120;小鼠腹水效價(jià)分別為1:64000、1:32000,c-myc單克隆抗體只與c-myc蛋白反應(yīng),與其他蛋白沒有交叉反應(yīng),這表明獲得的單克隆抗體具有較高的特異性和敏感性。本研究為腫瘤診斷試劑盒的研發(fā)提供了良好的技術(shù)基礎(chǔ)。
[Abstract]:Proto-oncogene c-myc is one of the important members of the myc family, and is the cell congener of v-myc, the oncogene of myeloid leukemia virus, which is located on chromosome 8q24.12-8q24.13. The molecular weight of c-myc protein encoded by the proto-oncogene is the function of 62kDa.c-myc gene.It is not only a translocative gene, but also a regulated gene which is regulated by a variety of substances. It can make cells proliferate infinitely, gain immortalized function and promote cell division. It is highly expressed in many tumors.Once the proto-oncogene of c-myc in normal cells is activated, the expression of c-myc mRNA and c-myc protein is increased abnormally, which makes the cells have high proliferative potential without the regulation of normal growth.Transformation to malignant phenotype, inhibition of cell differentiation, promotion of cell malignancy, and eventually tumorigenesis.In view of the highly positive correlation between overexpression of c-myc and carcinogenesis, c-myc antiserum can be used in the diagnosis of tumor. In this study, we prepared monoclonal antibodies against c-myc to provide basic experimental data for the development of tumor diagnostic kit.Three healthy female BALB/c mice were immunized with prokaryotic expression and purified c-myc protein antigen. The spleen cells were fused with SP2/0 myeloma cells at 10:1 ratio after three times immunization. Then the cells were selected by HAT selective medium and detected by indirect ELISA method.Two hybridoma cell lines, named SHH-1H11 and SHH-2A9, were obtained, which could secrete monoclonal antibody against c-myc stably.The identification of antibody subtypes showed that SHH-1H11H11 SHH-2A9 was IgG1 subclass and IgM subgroup respectively.BCA Protein Assay Reagent Kit showed that the antibody concentrations of SHH-1H11 supernatant and ascites were 0.47mg/ml and 2.51mg / ml SHH-2A9, respectively. The results of 0.49mg/ml and 2.45mg/ml.ELISA showed that the antibody concentration of SHH-1H11 supernatant and ascitic fluid were 0.49mg/ml and 2.45mg/ml.ELISA, respectively.The supernatant titers of the two hybridomas were 1: 10240 and 1: 5120, respectively, and the mice ascites titers were 1: 64000 and 1: 32000.The monoclonal antibodies against c-myc reacted only with c-myc protein and did not cross with other proteins, which indicated that the obtained monoclonal antibodies had high specificity and sensitivity.This study provides a good technical basis for the development of tumor diagnostic kit.
【學(xué)位授予單位】：遼寧師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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