【摘要】：目的:應(yīng)用基因工程技術(shù)制備腫瘤源性重組EPF,將其與弗氏佐劑混合后對BALB/c小鼠進行免疫,并以較為成熟的單克隆抗體雜交瘤技術(shù),建立分泌抗EPF單克隆抗體的雜交瘤細胞株,制備EPF單克隆抗體,為進一步探索以EPF單克隆抗體為基礎(chǔ)的酶聯(lián)免疫吸附測定(ELISA)等方法進行超早孕及某些惡性腫瘤等的早期診斷奠定基礎(chǔ)。方法:從黑色素瘤標(biāo)本中提取總RNA,RT-PCR擴增EPF基因,將擴增產(chǎn)物和載體pGEX-5X-1進行雙酶切后回收,連接載體和目的片段,獲得重組質(zhì)粒,將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21。在大腸桿菌BL21中通過IPTG進行誘導(dǎo)表達,然后用谷胱甘肽-瓊脂糖球珠親和層析純化,經(jīng)Xa酶切得到純的EPF蛋白。用SDS-PAGE鑒定其純度,用玫瑰花環(huán)抑制試驗(RIT)檢測其生物學(xué)活性。將制備的重組腫瘤源性EPF蛋白作為抗原免疫BALB/c小鼠,用免疫后的小鼠脾細胞與同系小鼠骨髓瘤細胞(NS-1)融合,并通過間接ELISA方法篩選克隆陽性雜交瘤細胞株,經(jīng)克隆化,獲得可穩(wěn)定分泌抗EPF單克隆抗體的細胞株。注入BALB/c小鼠腹腔制備腹水型單克隆抗體,經(jīng)Protein-G親和層析純化,SDS-PAGE和Western-blot等方法鑒定EPF單克隆抗體。結(jié)果:制備的重組EPF純度較好,具有良好的免疫原性。融合后經(jīng)克隆化獲得五株穩(wěn)定分泌抗EPF抗體的細胞株,將細胞注射入BALB/c小鼠腹腔獲得腹水型單克隆抗體,以親和層析法純化,SDS-PAGE分析顯示純化后去掉大部分雜蛋白,抗體純度較高,Western-blot分析顯示抗體與抗原匹配性良好。結(jié)論:制備的重組EPF蛋白純度高且具有良好的免疫原性。獲得五株穩(wěn)定分泌抗EPF抗體的雜交瘤細胞株。制備的EPF單克隆抗體純度好,與抗原匹配性良好。
[Abstract]:Objective: to prepare tumor-derived recombinant EPF, with Freund's adjuvant and immunize BALB/c mice with mature monoclonal antibody hybridoma technique. The hybridoma cell line secreting anti-EPF monoclonal antibody was established and the monoclonal antibody against EPF was prepared. In order to further explore the EPF monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) and other methods for the early diagnosis of ultra-early pregnancy and some malignant tumors and so on. Methods: total RNA,RT-PCR was extracted from melanoma specimens to amplify EPF gene. After double enzyme digestion of the amplified product and vector pGEX-5X-1, the recombinant plasmid was obtained by ligating the vector and the target fragment, and the recombinant plasmid was transformed into E. coli BL21.. E. coli BL21 was induced by IPTG and purified by glutathione-agarose beads affinity chromatography. The purified EPF protein was obtained by Xa digestion. Its purity was determined by SDS-PAGE and its biological activity was detected by rosette inhibition test (RIT). The recombinant tumor-derived EPF protein was used as antigen to immunize BALB/c mice. The spleen cells of immunized mice were fused with homologous mouse myeloma cells (NS-1), and the cloned hybridoma cell lines were screened by indirect ELISA. After cloning, the cell lines stably secreting anti-EPF monoclonal antibodies were obtained. The ascites monoclonal antibodies were prepared by intraperitoneal injection of BALB/c mice. The monoclonal antibodies against EPF were purified by Protein-G affinity chromatography, and identified by SDS-PAGE and Western-blot. Results: the prepared recombinant EPF had good purity and immunogenicity. After fusion, five cell lines stably secreting anti-EPF antibodies were obtained. The cells were injected into the peritoneal cavity of BALB/c mice to obtain ascites monoclonal antibodies, and purified by affinity chromatography. SDS-PAGE analysis showed that most of the miscellaneous proteins were removed after purification. The purity of the antibody was high, and Western-blot analysis showed that the antibody matched well with the antigen. Conclusion: the recombinant EPF protein has high purity and good immunogenicity. Five hybridoma cell lines stably secreting anti-EPF antibody were obtained. The EPF monoclonal antibody prepared is of good purity and good matching with the antigen.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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