本文選題：MUC1 + 重組MUC1融合蛋白�。� 參考：《吉林大學(xué)》2007年碩士論文

【摘要】： MUC1粘蛋白因其在多種腺癌包括乳腺癌、卵巢癌、肺癌等細(xì)胞上的高度異常表達(dá)使其成為腫瘤診斷和治療的有效靶點(diǎn)。研究發(fā)現(xiàn),腺癌患者尤其乳腺癌患者外周血中MUC1的水平較正常人高。其水平升高可以作為腫瘤輔助診斷指標(biāo)。但目前因方法各異,在臨床上尚未形成一種成熟的敏感特異的技術(shù)手段,而國(guó)外研發(fā)商業(yè)化生產(chǎn)的雙抗體夾心ELISA法(CASA test)對(duì)腺癌的檢出陽(yáng)性率較低。 本研究為檢測(cè)腫瘤患者外周血中MUC1蛋白水平,建立一種更加敏感的雙抗體夾心ELISA技術(shù)。首先構(gòu)建pGEX- MUC1和pMAL-MUC1兩種表達(dá)載體,轉(zhuǎn)化大腸桿菌,通過(guò)IPTG誘導(dǎo)MUC1-GST及MUC1-MBP融合蛋白在大腸桿菌中的表達(dá);經(jīng)Glutathione Sepharose 4B親和層析和非變性聚丙烯酰胺凝膠電泳純化MUC1-GST融合蛋白,通過(guò)Amylose Resin親和層析純化MUC1-MBP融合蛋白,經(jīng)Western blot鑒定蛋白的正確性;然后,將純化的MUC1-GST和MUC1-MBP融合蛋白分別免疫家兔和大鼠,制備兩種抗人MUC1多克隆抗體。最后,通過(guò)不同組合篩選合適的一級(jí)抗體、二級(jí)抗體及標(biāo)準(zhǔn)品,建立MUC1蛋白的雙抗體夾心ELISA檢測(cè)方法,采用雙抗體夾心ELISA方法檢測(cè)乳腺癌患者血清中MUC1蛋白含量。 本研究所建立的雙抗體夾心ELISA檢測(cè)血清中MUC1的方法,具有簡(jiǎn)便、快捷、敏感性強(qiáng)及檢出率高等優(yōu)點(diǎn),可望發(fā)展成為臨床上檢測(cè)腺癌的一種常規(guī)試劑盒。
[Abstract]:Due to its highly abnormal expression in many adenocarcinoma cells, including breast, ovarian, lung cancer, MUC1 mucin has become an effective target for tumor diagnosis and treatment. The level of MUC1 in peripheral blood of adenocarcinoma patients, especially breast cancer patients, was higher than that of normal controls. The elevated level can be used as an auxiliary diagnostic index of tumor. However, due to different methods, a mature sensitive and specific technique has not yet been developed in clinic, and the positive rate of detection of adenocarcinoma by developed and commercialized double antibody sandwich ELISA test is low. In order to detect the level of MUC1 protein in peripheral blood of tumor patients, a more sensitive sandwich ELISA technique with double antibodies was established. Firstly, two expression vectors, pGEX- MUC1 and pMAL-MUC1, were constructed and transformed into Escherichia coli, and MUC1-GST and MUC1-MBP fusion proteins were induced to express in E. coli by IPTG. MUC1-GST fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and non-denaturing polyacrylamide gel electrophoresis. The MUC1-MBP fusion protein was purified by Amylose Resin affinity chromatography and identified by Western blot. Then the purified MUC1-GST and MUC1-MBP fusion proteins were immunized with rabbit and rat respectively to prepare two kinds of anti-human MUC1 polyclonal antibodies. Finally, by screening suitable primary, secondary and standard antibodies in different combinations, a double antibody sandwich ELISA method for detection of MUC1 protein was established, and the content of MUC1 protein in serum of breast cancer patients was detected by double antibody sandwich ELISA method. The method of double antibody sandwich ELISA for detection of MUC1 in serum is simple, rapid, sensitive and high detection rate. It is expected to develop into a routine kit for the detection of adenocarcinoma in clinic.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

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