抗-URG11和抗-URG4單克隆抗體的制備及初步鑒定
發(fā)布時間：2018-09-17 17:42
【摘要】： 【背景】urg11(up regulated gene 11)和urg4(up regulated gene 4)是近期發(fā)現(xiàn)的可以被乙肝病毒X(hepatitis B Virus x, HBx)蛋白上調(diào)的兩個新基因。研究表明[1, 2]urg11不但可以增強細胞體外增殖能力、促進細胞在軟瓊脂中非錨定生長和加快裸鼠體內(nèi)成瘤,而且還可以通過上調(diào)β-連接蛋白(β-catenin)的表達參與肝細胞癌的發(fā)生。urg4 [3, 4]不僅可以增強細胞體外增殖能力、促進細胞在軟瓊脂中非錨定生長和加快裸鼠體內(nèi)成瘤,而且還可以促進肝癌的形成和胃癌的發(fā)生。更重要的是urg11和urg4聯(lián)合其它新克隆的基因可以提前3年預警肝癌的發(fā)生[5]。因此,進一步研究其功能具有重要的科學價值,但目前尚未有可用的針對URG11或URG4的單克隆抗體。 【目的】通過人工合成多肽片段制備抗-URG11和抗-URG4單克隆抗體,并對所制備的單克隆抗體進行初步鑒定。 【方法】(1)根據(jù)已發(fā)表的文獻[1,3]通過人工合成多肽,并將此多肽分別連接鑰孔戚血藍素(簡稱:peptide-KLH)或牛血清蛋白(簡稱:peptide-BSA);(2)以peptide-KLH作為抗原免疫BALB/c小鼠;(3)取血清效價大于1:10000的小鼠的脾細胞與骨髓瘤細胞SP2/0進行融合,以peptide-BSA包被,通過酶聯(lián)免疫吸附實驗(ELISA)檢測雜瘤細胞的上清進行篩選;陽性孔的雜交瘤細胞予保留,用有限稀釋法進行三次克隆化,得到能穩(wěn)定分泌單克隆抗體的雜交瘤細胞株;(4)應用所得的雜交瘤細胞株通過腹腔注射BALB/c小鼠以大量生產(chǎn)單克隆抗體;(5)通過鼠單克隆抗體亞類分型試劑盒鑒定單克隆抗體的亞類,采用蛋白質(zhì)印跡法(Western blotting)和免疫組化方法證明新制備的單克隆抗體能識別目的蛋白。 【結(jié)果】(1)得到一株能穩(wěn)定分泌抗-URG11單克隆抗體的雜交瘤細胞株(3D2);應用蛋白質(zhì)印跡法和免疫組化方法證明新制備的抗-URG11單克隆抗體能識別天然的URG11;此單克隆抗體的亞類為IgG2a/κ。(2)得到一株能穩(wěn)定分泌抗-URG4單克隆抗體的雜交瘤細胞株(1A8);其分泌的單克隆抗體的亞類為IgG2a/κ;通過蛋白質(zhì)印跡法和免疫組化方法證實新制備的抗-URG4單克隆抗體能識別天然的URG4。 【結(jié)論】兩株雜交瘤細胞株(3D2和1A8)都能穩(wěn)定分泌目的單克隆抗體;蛋白印跡法和免疫組化方法證明新制備的兩種單克隆抗體能分別識別自己天然的目的蛋白(URG11或URG4),為更好地研究urg11和urg4的生物學功能奠定了基礎。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
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