利用DNA免疫技术制备ASB11蛋白的多克隆抗体

ASB11参与胚胎神经祖细胞的发育、再生性肌发生以及泛素化等过程,但其机制仍不清楚。为了进一步研究斑马鱼Asb11基因的作用机制,本研究采用DNA免疫技术制备了ASB11多克隆抗体；利用斑马鱼Asb11的cDNA构建pCAGGS-P7/ASB11重组表达质粒,肌肉注射入6-8周龄的BALB/c小鼠体内,诱导抗原蛋白的表达和免疫应答的发生。结果显示,制备的pCAGGS-P7/ASB11重组质粒具有较好的免疫原性；将提取的抗血清进行Western-blot和免疫荧光检测,显示所制备的多克隆抗体抗体效价为1∶400,抗血清抗体能特异的结合ASB11蛋白。本研究为后续的功能研究奠定了基础。

Abstract

ASB11 is involved in the development of embryonic neural progenitor cells, regenerative myogenesis and ubiquitination, but its detailed mechanism of action has not been known. In order to further analyze the mechanism of zebrafish Asb11 gene, the anti-ASB11 specific polyclonal antibody by immunizing mice with DNA was prepared. The eukaryotic expression vector pCAGGS-P7/ASB11 was constructed by using cDNA of zebrafish Asb11. BALB/c mice of 6-8 weeks old were immunized with pCAGGS-P7/ASB11 to prepare anti-serum against ASB11. The results showed that the pCAGGS-P7/ASB11 recombinant plasmid was constructed with a rather good immunogenicity. The antibody valence of anti-serum obtained from the immunized mice was 1∶400. Western blotting and indirect immunofluorescence showed that the anti-serum prepared by immunizing mice with DNA could specifically recognize the ASB11 protein. This research established afoundation to reveal the mechanism of ASB11 protein.

参考文献

[1] HILTON D J, RICHARDSON R T, ALEXANDER W S, et al. Twenty proteins containing a C-terminal SOCS box form five structural classes［J］. Proceedings of the National Academy of Sciences USA, 1998, 95(1):114-119.

[2] KILE B T, METCALF D, MIFSUD S, et al. Functional analysis of Asb-1 using genetic modification in mice［J］. Molecular and Cellular Biology, 2001, 21(18):6189-6197.

[3] CHUNG A S, GUAN Y J, YUAN Z L, et al. Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II［J］. Molecular and Cellular Biology, 2005, 25(11):4716-4726.

[4] SEALE P, ISHIBASHI J, HOLTERMAN C, et al. Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell［J］. Developmental Biology, 2004, 275(2):287-300.

[5] SCHIFFER J M, MALMSTROM R D, PARNELL J, et al. Model of the ankyrin and SOCS box protein, ASB9, E3 ligase reveals a mechanism for dynamic ubiquitin transfer［J］. Structure, 2016, 24(8):1248-1256.

[6] DIKS S H, BINK R J, VAN D E WATER S, et al. The novel gene asb11:a regulator of the size of the neural progenitor compartment［J］. The Journal of Cell Biology, 2006, 174(4):581 -592.

[7] DIKS S H, SARTORI DA SILVA MA, HILLEBRANDS J, et al. d-Asb11 is an essential mediator of canonical Delta-Notch signaling［J］. Nature Cell Biology, 2008, 10(10):1190-1198.

[8] SARTORI D S M, TEE J M, PARIDAEN J, et al. Essential role for the d-Asb11 cul5 box domain for proper notch signaling and neural cell fate decisions in vivo［J］. PLoS One, 2010, 5(11):e14023.

[9] TEE J M, SARTORI D S M, RYGIEL A M, et al. Asb11 is a regulator of embryonic and adult regenerative myogenesis［J］. Stem Cells and Development, 2012, 21(17):3091-3103.

[10] 胡君健. 肿瘤发生候选基因ASB11-1和心脏发育候选基因XIRP1的表达和功能初步研究［D］. 长沙:湖南师范大学, 2008.

HU Junjian. Expression and functional analysis of tumorigenesis ASB11-1 and cardiogenesis XIRP1［D］. Changsha:Hunan Normal University, 2008.

[11] 杨建华. 人类ASB11基因在肌细胞分化中的调控作用［D］. 长沙:湖南师范大学, 2008.

YANG Jianhua. Role of human ASB11 in myogenesis［D］. Changsha:Hunan Normal University, 2008.

[12] 孟和宝力高, 其其格, 额尔敦. DNA免疫的作用机制及影响因素［J］. 细胞与分子免疫学杂志, 2007, 23(7):682.

MengHebaoligao, QI Qige, E Erdun. The mechanism of action and influence of DNA immunization［J］.Chinese Journal of Cellular and Molecular Immunology, 2007, 23(7): 682.

[13] WOLFF J A, DOWTY M E, JIAO S, et al. Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle［J］. Journal of Cell Science, 1992, 103(4): 1249-1259.

[14] 刘淑颖, 陈雨欣, 王世霞, 等. DNA免疫技术在高质量单克隆抗体诱导开发中的应用进展［J］. 药学进展, 2017, 09(41):662-669.

LIU Shuyin, CHEN Yuxin, WANG Shixia, et al. Progress in application of DNA immunization technique in R&D of high-quality monoclonal antibodies［J］. Progress Pharmaceutical Sciences, 2017, 09(41):662-669.

[15] 王和勇, 向阳, 唐亮, 等. 不同部位皮下注射DNA疫苗对小鼠体液免疫的影响［J］. 西南大学学报, 2008, 30(10):89-94.

WANG Heyong, XIANG Yang, TANG Liang, et al. Effect of subcutaneous injection of DNA vaccine at different sites on humoral immunity in mice［J］. Journal of Southwest University, 2008, 30(10):89-94.

[16] 楚琰, 吴兴安. DNA疫苗及其免疫途径的研究进展［J］. 中国医药生物技术, 2010, 5(04):285-288.

CHU Yan, WU Xingan. Research progress of DNA vaccine and it’s immune pathway［J］. Chinese Medicinal Biotechnology, 2010, 5(04):285-288.

[17] 姜永萍, 张洪波, 步志高, 等. 表达载体pCAGGS显著增强禽流感DNA疫苗的免疫保护效果［J］. 中国农业科学, 2006, 39(4):825-830.

JIANG Yongping, ZHANG Hongbo, BU Zhigao, et al. Enhanced protective efficacy of avian influenza DNA vaccine with expressive vector pCAGGS［J］. Scientia Agricultura Sinica, 2006, 39(4):825-830.

[18] 刘丽梅, 陈宗涛, 高娜, 等. 利用DNA免疫技术制备抗登革2型病毒NS2B蛋白多克隆抗体［J］. 第三军医大学学报, 2010, 32(4):327-330.

LIU Limei, CHEN Zongtao, GAO Na, et al. Preparation of polyclonal antibodies against NS2B of dengue virus serotype 2 by immunization with DNA［J］. Acta Academiae Medicinae Militaris Tertiae, 2010, 32(4):327-330.

[19] WOLFF J A, MALONE R W, WILLIAMS P, et al. Direct gene transfer into mouse muscle in vivo［J］. Science, 1990, 247(4949):1465-1468.

[20] TOKUOKA M, MIYOSHI N, HITORA T, et al. Clinical significance of ASB9 in human colorectal cancer［J］. International Journal of Oncology, 2010, 37(5): 1105-1111.

蔡英桂, 尹丽阳, 王宏波, 陈宇, 曾蓉, 吴秀山, 叶湘漓. 利用DNA免疫技术制备ASB11蛋白的多克隆抗体[J]. 激光生物学报, 2018, 27(3): 260. CAI Yinggui, YIN Liyang, WANG Hongbo, CHEN Yu, ZENG Rong, WU Xiushan, YE Xiangli. Preparation of Polyclonal Antibody Against ASB11 Protein by DNA Immunization[J]. Acta Laser Biology Sinica, 2018, 27(3): 260.

利用DNA免疫技术制备ASB11蛋白的多克隆抗体

关于本站 Cookie 的使用提示

全站搜索

利用DNA免疫技术制备ASB11蛋白的多克隆抗体

相关论文

相关资讯

关于本站 Cookie 的使用提示

全站搜索