肿瘤源性早孕因子单克隆抗体制备及鉴定
发布时间：2019-03-20 17:57
【摘要】：目的:应用基因工程技术制备肿瘤源性重组EPF,将其与弗氏佐剂混合后对BALB/c小鼠进行免疫,并以较为成熟的单克隆抗体杂交瘤技术,建立分泌抗EPF单克隆抗体的杂交瘤细胞株,制备EPF单克隆抗体,为进一步探索以EPF单克隆抗体为基础的酶联免疫吸附测定(ELISA)等方法进行超早孕及某些恶性肿瘤等的早期诊断奠定基础。方法:从黑色素瘤标本中提取总RNA,RT-PCR扩增EPF基因,将扩增产物和载体pGEX-5X-1进行双酶切后回收,连接载体和目的片段,获得重组质粒,将重组质粒转化大肠杆菌BL21。在大肠杆菌BL21中通过IPTG进行诱导表达,然后用谷胱甘肽-琼脂糖球珠亲和层析纯化,经Xa酶切得到纯的EPF蛋白。用SDS-PAGE鉴定其纯度,用玫瑰花环抑制试验(RIT)检测其生物学活性。将制备的重组肿瘤源性EPF蛋白作为抗原免疫BALB/c小鼠,用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合,并通过间接ELISA方法筛选克隆阳性杂交瘤细胞株,经克隆化,获得可稳定分泌抗EPF单克隆抗体的细胞株。注入BALB/c小鼠腹腔制备腹水型单克隆抗体,经Protein-G亲和层析纯化,SDS-PAGE和Western-blot等方法鉴定EPF单克隆抗体。结果:制备的重组EPF纯度较好,具有良好的免疫原性。融合后经克隆化获得五株稳定分泌抗EPF抗体的细胞株,将细胞注射入BALB/c小鼠腹腔获得腹水型单克隆抗体,以亲和层析法纯化,SDS-PAGE分析显示纯化后去掉大部分杂蛋白,抗体纯度较高,Western-blot分析显示抗体与抗原匹配性良好。结论:制备的重组EPF蛋白纯度高且具有良好的免疫原性。获得五株稳定分泌抗EPF抗体的杂交瘤细胞株。制备的EPF单克隆抗体纯度好,与抗原匹配性良好。
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2444466
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前6条
1 周泉,易传祝,郭海萍,王家骥;抗早孕因子单克隆抗体的制备与鉴定[J];免疫学杂志;2005年S1期
2 张冬云,王家骥,杨英,林丽白,冯苏妹,苏宝田;肿瘤源性早孕因子的筛检、纯化及鉴定[J];中国生化药物杂志;2002年01期
3 杨德吉，吉传义，龚晓明;早孕因子化学本质的研究进展[J];生命的化学(中国生物化学会通讯);1995年01期
4 张冬云,王家骥,林丽白,张莉;“人流血”中早孕因子的分离、纯化及鉴定[J];中国生物制品学杂志;2000年03期
5 王敏华,苏宝田;滋养细胞肿瘤患者血清早孕因子样物质的检测[J];实用癌症杂志;1993年01期
6 李世新,武小文;早孕因子对流产预后的预测研究[J];现代诊断与治疗;1999年S1期
，本文编号：2444466
本文链接：https://www.wllwen.com/xiyixuelunwen/2444466.html
最近更新
教材专著
