人突变CD59蛋白的提取纯化与单克隆抗体的制备
发布时间：2018-08-25 19:11
【摘要】： 目的建立转染人突变CD59(hmCD59)-pALTER-MAX重组质粒的CHO细胞模型；利用ANTI-FLAG M2亲和层析的方法提取纯化hmCD59重组蛋白；以纯化的hmCD59重组蛋白免疫Balb／c小鼠，制备抗hmCD59的多克隆及单克隆抗体；为最终设计出以抗hmCD59单克隆抗体为基础的诊断与治疗措施打下坚实的理论基础，为糖尿病血管并发症的研究提供新的思路与手段。 方法将含有hmCD59全长序列的重组pALTER-MAX质粒，运用脂质体介导法，与pcDNA3质粒共转染中国仓鼠卵巢细胞(CHO细胞)，，以新霉素类似物G418筛选出抗性克隆；采用免疫荧光技术筛选转染细胞阳性克隆；不连续聚丙烯酰胺凝胶电泳(SDS-PAGE)检测转染CHO细胞hmCD59的表达情况；表达产物经ANTI-FLAG M2 affinity gel亲和层析纯化，以SDS-PAGE、免疫印迹法(Western blot)和酶联免疫吸附实验(ELISA)对纯化产物进行鉴定；将纯化的hmCD59重组蛋白免疫Balb／c小鼠，分别制备抗hmCD59的多克隆及单克隆抗体，ELISA法检测抗体效价及免疫学活性。 结果免疫荧光与SDS-PAGE检测证实建立了表达hmCD59的CHO转染细胞模型；经ANTI-FLAG M2 affinity gel亲和层析获得电泳纯的hmCD59，蛋白浓度为0.6mg／ml，Western blot与ELISA检测结果显示纯化hmCD59与抗hmCD59抗体特异结合；纯化hmCD59免疫Balb／c小鼠获得的抗血清间接ELISA效价为1∶100；通过淋巴细胞杂交瘤技术获得1株分泌特异性单克隆抗体的杂交瘤细胞株，其培养上清效价为1∶16。 结论成功构建了转染hmCD59-pALTER-MAX重组质粒的CHO细胞模型；重组hmCD59在CHO细胞表面稳定表达；亲和层析法可以获得电泳纯并具有免疫学活性的hmCD59蛋白；进而免疫Balb／c小鼠获得了抗hmCD59的多克隆及单克隆抗体，为抗hmCD59抗体的功能研究及临床应用奠定了基础。
[Abstract]:Objective to establish a CHO cell model transfected with human mutant CD59 (hmCD59) -pALTER-MAX recombinant plasmid, extract and purify hmCD59 recombinant protein by ANTI-FLAG M2 affinity chromatography, immunize Balb/c mice with purified hmCD59 recombinant protein, and prepare anti-hmCD59 polyclonal and monoclonal antibodies. It provides a solid theoretical basis for the design of diagnostic and therapeutic measures based on monoclonal antibodies against hmCD59, and provides new ideas and means for the study of diabetic vascular complications. Methods Recombinant pALTER-MAX plasmid containing the full-length hmCD59 sequence was co-transfected with pcDNA3 plasmid into Chinese hamster ovarian cells (CHO cells) by liposome mediated method. The resistant clones were screened by neomycin analogues G418. The positive clones were screened by immunofluorescence technique, the expression of hmCD59 in transfected CHO cells was detected by discontinuous polyacrylamide gel electrophoresis (SDS-PAGE), and the expression product was purified by ANTI-FLAG M2 affinity gel affinity chromatography. SDS-PAGE, immunoblot assay (Western blot) and enzyme-linked immunosorbent assay (ELISA) were used to identify the purified product, and the purified hmCD59 recombinant protein was used to immunize Balb/c mice. The polyclonal anti-hmCD59 and monoclonal antibody were prepared to detect the antibody titer and immunological activity by Elisa. Results the CHO transfected cell model expressing hmCD59 was established by immunofluorescence and SDS-PAGE detection, and the concentration of hmCD59, protein was 0.6 mg / ml ~ (-1) by ANTI-FLAG M2 affinity gel affinity chromatography. The results showed that the purified hmCD59 specifically combined with anti-hmCD59 antibody. The indirect antiserum ELISA titer of purified hmCD59 immunized Balb/c mice was 1: 100, and a hybridoma cell line secreting specific monoclonal antibody was obtained by lymphocyte hybridoma technique, the titer of culture supernatant was 1: 16. Conclusion the CHO cell model transfected with hmCD59-pALTER-MAX recombinant plasmid was successfully constructed, the recombinant hmCD59 was stably expressed on the surface of CHO cells, and the purified hmCD59 protein with immunological activity could be obtained by affinity chromatography. The polyclonal and monoclonal antibodies against hmCD59 were obtained by immunizing Balb/c mice, which laid a foundation for the functional study and clinical application of anti hmCD59 antibodies.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2203831
[Abstract]:Objective to establish a CHO cell model transfected with human mutant CD59 (hmCD59) -pALTER-MAX recombinant plasmid, extract and purify hmCD59 recombinant protein by ANTI-FLAG M2 affinity chromatography, immunize Balb/c mice with purified hmCD59 recombinant protein, and prepare anti-hmCD59 polyclonal and monoclonal antibodies. It provides a solid theoretical basis for the design of diagnostic and therapeutic measures based on monoclonal antibodies against hmCD59, and provides new ideas and means for the study of diabetic vascular complications. Methods Recombinant pALTER-MAX plasmid containing the full-length hmCD59 sequence was co-transfected with pcDNA3 plasmid into Chinese hamster ovarian cells (CHO cells) by liposome mediated method. The resistant clones were screened by neomycin analogues G418. The positive clones were screened by immunofluorescence technique, the expression of hmCD59 in transfected CHO cells was detected by discontinuous polyacrylamide gel electrophoresis (SDS-PAGE), and the expression product was purified by ANTI-FLAG M2 affinity gel affinity chromatography. SDS-PAGE, immunoblot assay (Western blot) and enzyme-linked immunosorbent assay (ELISA) were used to identify the purified product, and the purified hmCD59 recombinant protein was used to immunize Balb/c mice. The polyclonal anti-hmCD59 and monoclonal antibody were prepared to detect the antibody titer and immunological activity by Elisa. Results the CHO transfected cell model expressing hmCD59 was established by immunofluorescence and SDS-PAGE detection, and the concentration of hmCD59, protein was 0.6 mg / ml ~ (-1) by ANTI-FLAG M2 affinity gel affinity chromatography. The results showed that the purified hmCD59 specifically combined with anti-hmCD59 antibody. The indirect antiserum ELISA titer of purified hmCD59 immunized Balb/c mice was 1: 100, and a hybridoma cell line secreting specific monoclonal antibody was obtained by lymphocyte hybridoma technique, the titer of culture supernatant was 1: 16. Conclusion the CHO cell model transfected with hmCD59-pALTER-MAX recombinant plasmid was successfully constructed, the recombinant hmCD59 was stably expressed on the surface of CHO cells, and the purified hmCD59 protein with immunological activity could be obtained by affinity chromatography. The polyclonal and monoclonal antibodies against hmCD59 were obtained by immunizing Balb/c mice, which laid a foundation for the functional study and clinical application of anti hmCD59 antibodies.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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