鸡源p53单克隆抗体的制备和鉴定
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摘要
转录因子p53广泛参与调节机体生理和病理过程,包括生长发育、新陈代谢、免疫应答、肿瘤发生、发展等,但是禽类p53的生物学功能尚不清楚。本研究扩增鸡p53基因并连入p ET-30a原核表达载体,将载体转化至大肠杆菌BL21中,利用IPTG诱导表达p53蛋白。经SDS-PAGE分离,收集p53蛋白并免疫BALB/c小鼠,制备多克隆抗体。取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,利用免疫印记、间接免疫荧光和酶联免疫吸附法筛选阳性细胞株。共获得6株稳定分泌鸡p53蛋白单克隆抗体的杂交瘤细胞株,分别命名为1D7、3C4、4D4、4F6、4F7和4H2,均为Ig G亚类,并鉴定了1D7的特异性。本研究成功实现了鸡源p53蛋白的重组表达,制备出特异性较好的抗体,为研究p53在鸡体内的生物学作用奠定了基础。
Transcription factor p53 has been shown to be involved in regulating physiological and pathogenic processes, including growth and development, metabolism, immune response and tumor development. But poultry p53 function is still not clear. This study aimed to prepare monoclonal antibodies against chicken p53. The p53 gene was amplified and cloned into the p ET-30 a vector. The recombinant prokaryotic expression plasmid was transformed into BL21 and induced by IPTG express p53 which was then analyzed by SDS-PAGE. The p53 protein was collected and used to immunize Balb/c mice to generate polyclonal antibodies against chicken p53 protein. In addition, spleen cells from immunized mice were fused with murine myeloma SP2/0 cells. Hybridomas secreting specific MAb were screened by Western blot(WB), indirect immunofluorescence assay(IFA), and enzyme-linked immunosorbent assay(ELISA). As a result, six positive hybridoma cell lines were identified and designated 1 D7, 3 C4, 4 D4, 4 F6, 4 F7 and 4 H2, the isotype of which was Ig G,1 D7 specificity was identified. These indicated that p53 could expressed in E. coli BL21(DE3), and the specific monoclonal antibodies against p53 were successfully generated, which laid a foundation for further study on p53 in chicken.
Transcription factor p53 has been shown to be involved in regulating physiological and pathogenic processes, including growth and development, metabolism, immune response and tumor development. But poultry p53 function is still not clear. This study aimed to prepare monoclonal antibodies against chicken p53. The p53 gene was amplified and cloned into the p ET-30 a vector. The recombinant prokaryotic expression plasmid was transformed into BL21 and induced by IPTG express p53 which was then analyzed by SDS-PAGE. The p53 protein was collected and used to immunize Balb/c mice to generate polyclonal antibodies against chicken p53 protein. In addition, spleen cells from immunized mice were fused with murine myeloma SP2/0 cells. Hybridomas secreting specific MAb were screened by Western blot(WB), indirect immunofluorescence assay(IFA), and enzyme-linked immunosorbent assay(ELISA). As a result, six positive hybridoma cell lines were identified and designated 1 D7, 3 C4, 4 D4, 4 F6, 4 F7 and 4 H2, the isotype of which was Ig G,1 D7 specificity was identified. These indicated that p53 could expressed in E. coli BL21(DE3), and the specific monoclonal antibodies against p53 were successfully generated, which laid a foundation for further study on p53 in chicken.
引文
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[4]Serrano M,Lin A W,Mccurrach M E,et al.Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a[J].Cell,1997,88(5):593-602.
[5]Sugrue M M,Shin D Y,Lee S W,et al.Wild-type p53triggers a rapid senescence program in human tumor cells lacking functional p53[J].Proc Natl Acad Sci U S A,1997,94(18):9648-9653.
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[7]Gasco M,Yulug I G,Crook T.TP53 mutations in familial breast cancer:functional aspects[J].Hum Mutat,2003,21(3):301-306.
[8]Qian Y,Chen X.Tumor suppression by p53:making cells senescent[J].Histol Histopathol,2010,25(4):515-526.
[9]陈洪菊,屈艺,母得志.m TOR信号通路的生物学功能[J].生命的化学,2010(4):555-561.
[10]Jing K,Song K S,Shin S,et al.Docosahexaenoic acid induces autophagy through p53/AMPK/m TOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53[J].Autophagy,2011,7(11):1348-1358.
[11]Matissek K J,Mossalam M,Okal A,et al.The DNA binding domain of p53 is sufficient to trigger a potent apoptotic response at the mitochondria[J].Mol Pharm,2013,10(10):3592-3602.
[12]Wolff S,Erster S,Palacios G,et al.p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity[J].Cell Res,2008,18(7):733-744.
[13]Mihara M,Erster S,Zaika A,et al.p53 has a direct apoptogenic role at the mitochondria[J].Mol Cell,2003,11(3):577-590.
[14]Joerger A C,Fersht A R.The tumor suppressor p53:from structures to drug discovery[J].Cold Spring Harb Perspect Biol,2010,2(6):a000919.
[15]Prives C,Manfredi J J.The p53 tumor suppressor protein:meeting review[J].Genes Dev,1993,7(4):529-534.
[16]王志洁.p53基因结构与功能研究的新进展[J].国际遗传学杂志,1999(1):25-29.
[17]Thut C J,Chen J L,Klemm R,et al.p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60[J].Science,1995,267(5194):100-104.
[18]Kussie P H,Gorina S,Marechal V,et al.Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain[J].Science,1996,274(5289):948-953.