抗-URG11和抗-URG4单克隆抗体的制备及初步鉴定
发布时间：2018-09-17 17:42
【摘要】： 【背景】urg11(up regulated gene 11)和urg4(up regulated gene 4)是近期发现的可以被乙肝病毒X(hepatitis B Virus x, HBx)蛋白上调的两个新基因。研究表明[1, 2]urg11不但可以增强细胞体外增殖能力、促进细胞在软琼脂中非锚定生长和加快裸鼠体内成瘤,而且还可以通过上调β-连接蛋白(β-catenin)的表达参与肝细胞癌的发生。urg4 [3, 4]不仅可以增强细胞体外增殖能力、促进细胞在软琼脂中非锚定生长和加快裸鼠体内成瘤,而且还可以促进肝癌的形成和胃癌的发生。更重要的是urg11和urg4联合其它新克隆的基因可以提前3年预警肝癌的发生[5]。因此,进一步研究其功能具有重要的科学价值,但目前尚未有可用的针对URG11或URG4的单克隆抗体。 【目的】通过人工合成多肽片段制备抗-URG11和抗-URG4单克隆抗体,并对所制备的单克隆抗体进行初步鉴定。 【方法】(1)根据已发表的文献[1,3]通过人工合成多肽,并将此多肽分别连接钥孔戚血蓝素(简称:peptide-KLH)或牛血清蛋白(简称:peptide-BSA);(2)以peptide-KLH作为抗原免疫BALB/c小鼠;(3)取血清效价大于1:10000的小鼠的脾细胞与骨髓瘤细胞SP2/0进行融合,以peptide-BSA包被,通过酶联免疫吸附实验(ELISA)检测杂瘤细胞的上清进行筛选;阳性孔的杂交瘤细胞予保留,用有限稀释法进行三次克隆化,得到能稳定分泌单克隆抗体的杂交瘤细胞株;(4)应用所得的杂交瘤细胞株通过腹腔注射BALB/c小鼠以大量生产单克隆抗体;(5)通过鼠单克隆抗体亚类分型试剂盒鉴定单克隆抗体的亚类,采用蛋白质印迹法(Western blotting)和免疫组化方法证明新制备的单克隆抗体能识别目的蛋白。 【结果】(1)得到一株能稳定分泌抗-URG11单克隆抗体的杂交瘤细胞株(3D2);应用蛋白质印迹法和免疫组化方法证明新制备的抗-URG11单克隆抗体能识别天然的URG11;此单克隆抗体的亚类为IgG2a/κ。(2)得到一株能稳定分泌抗-URG4单克隆抗体的杂交瘤细胞株(1A8);其分泌的单克隆抗体的亚类为IgG2a/κ;通过蛋白质印迹法和免疫组化方法证实新制备的抗-URG4单克隆抗体能识别天然的URG4。 【结论】两株杂交瘤细胞株(3D2和1A8)都能稳定分泌目的单克隆抗体;蛋白印迹法和免疫组化方法证明新制备的两种单克隆抗体能分别识别自己天然的目的蛋白(URG11或URG4),为更好地研究urg11和urg4的生物学功能奠定了基础。
[Abstract]:[background] urg11 (up regulated gene 11) and urg4 (up regulated gene 4) are two novel genes that can be up-regulated by hepatitis B virus X (hepatitis B Virus x, HBx) protein. The results showed that [1,2] urg11 could not only enhance cell proliferation in vitro, promote cell growth in soft Agar, but also accelerate tumorigenesis in nude mice. Moreover, the expression of 尾 -catenin can be involved in the carcinogenesis of hepatocellular carcinoma. Urg4 [3,4] can not only enhance cell proliferation in vitro, promote cell growth in soft Agar, but also accelerate tumor formation in nude mice. It can also promote the formation of liver cancer and gastric cancer. More importantly, urg11 and urg4 combined with other newly cloned genes can forewarn liver cancer 3 years ahead of time [5]. Therefore, it is of great scientific value to further study its function. But there is no available monoclonal antibody against URG11 or URG4. [objective] to prepare anti-URG11 and anti-URG4 monoclonal antibodies by synthetic polypeptide fragments. The monoclonal antibody was preliminarily identified. [methods] (1) according to the published literature [1], the peptide was synthesized by artificial synthesis. The peptide was linked to the key peptide-KLH or the bovine serum protein (BSA); (2) to immunize BALB/c mice with peptide-KLH as antigen. (3) spleen cells of mice with serum titers greater than 1: 10000 were fused with SP2/0 of myeloma cells to be coated with peptide-BSA. The supernatants of the hybridoma cells were screened by Elisa (ELISA), and the hybridoma cells with positive pore were preserved and cloned three times by the limited dilution method. Hybridoma cell lines with stable secretion of monoclonal antibodies were obtained; (4) hybridoma cell lines were obtained by intraperitoneal injection of BALB/c mice for mass production of monoclonal antibodies; (5) subclasses of monoclonal antibodies were identified by mouse monoclonal antibody subclass typing kit. Western blot (Western blotting) and immunohistochemical methods were used to prove that the newly prepared monoclonal antibody could recognize the target protein. [results] (1) A monoclonal anti-URG11 antibody secreted stably was obtained. Using Western blot and immunohistochemistry, it was proved that the newly prepared anti-URG11 monoclonal antibody could recognize natural URG11;, the subclass of which was IgG2a/ 魏. (2) A monoclonal anti-URG4 monoclonal antibody could be secreted stably. The hybridoma cell line (1A8) secreted IgG2a/ 魏; the newly prepared monoclonal antibody against URG4 was proved to recognize natural URG4. by Western blot and immunohistochemistry. [conclusion] two hybridoma cell lines (3D2 and 1A8) can secrete monoclonal antibodies stably. Western blot and immunohistochemistry showed that the two new monoclonal antibodies could recognize their own natural target protein (URG11 or URG4) respectively, which laid a foundation for better study of the biological functions of urg11 and urg4.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2246647
[Abstract]:[background] urg11 (up regulated gene 11) and urg4 (up regulated gene 4) are two novel genes that can be up-regulated by hepatitis B virus X (hepatitis B Virus x, HBx) protein. The results showed that [1,2] urg11 could not only enhance cell proliferation in vitro, promote cell growth in soft Agar, but also accelerate tumorigenesis in nude mice. Moreover, the expression of 尾 -catenin can be involved in the carcinogenesis of hepatocellular carcinoma. Urg4 [3,4] can not only enhance cell proliferation in vitro, promote cell growth in soft Agar, but also accelerate tumor formation in nude mice. It can also promote the formation of liver cancer and gastric cancer. More importantly, urg11 and urg4 combined with other newly cloned genes can forewarn liver cancer 3 years ahead of time [5]. Therefore, it is of great scientific value to further study its function. But there is no available monoclonal antibody against URG11 or URG4. [objective] to prepare anti-URG11 and anti-URG4 monoclonal antibodies by synthetic polypeptide fragments. The monoclonal antibody was preliminarily identified. [methods] (1) according to the published literature [1], the peptide was synthesized by artificial synthesis. The peptide was linked to the key peptide-KLH or the bovine serum protein (BSA); (2) to immunize BALB/c mice with peptide-KLH as antigen. (3) spleen cells of mice with serum titers greater than 1: 10000 were fused with SP2/0 of myeloma cells to be coated with peptide-BSA. The supernatants of the hybridoma cells were screened by Elisa (ELISA), and the hybridoma cells with positive pore were preserved and cloned three times by the limited dilution method. Hybridoma cell lines with stable secretion of monoclonal antibodies were obtained; (4) hybridoma cell lines were obtained by intraperitoneal injection of BALB/c mice for mass production of monoclonal antibodies; (5) subclasses of monoclonal antibodies were identified by mouse monoclonal antibody subclass typing kit. Western blot (Western blotting) and immunohistochemical methods were used to prove that the newly prepared monoclonal antibody could recognize the target protein. [results] (1) A monoclonal anti-URG11 antibody secreted stably was obtained. Using Western blot and immunohistochemistry, it was proved that the newly prepared anti-URG11 monoclonal antibody could recognize natural URG11;, the subclass of which was IgG2a/ 魏. (2) A monoclonal anti-URG4 monoclonal antibody could be secreted stably. The hybridoma cell line (1A8) secreted IgG2a/ 魏; the newly prepared monoclonal antibody against URG4 was proved to recognize natural URG4. by Western blot and immunohistochemistry. [conclusion] two hybridoma cell lines (3D2 and 1A8) can secrete monoclonal antibodies stably. Western blot and immunohistochemistry showed that the two new monoclonal antibodies could recognize their own natural target protein (URG11 or URG4) respectively, which laid a foundation for better study of the biological functions of urg11 and urg4.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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