鸡细胞周期蛋白依赖性激酶抑制蛋白p21单克隆抗体的制备
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摘要
为制备特异性的鸡细胞周期蛋白依赖性激酶抑制蛋白p21单克隆抗体(mAb),利用反转录聚合酶链式反应(RT-PCR)扩增鸡p21基因,将其克隆入原核表达载体pET-30a中,随后转化进入BL21(DE3)中,利用异丙基硫代半乳糖苷(IPTG)诱导表达,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离。切胶研磨,将收集的鸡p21蛋白腹腔免疫BALB/c小鼠,三免后,选取抗体阳性的小鼠脾与小鼠骨髓瘤细胞SP2/0融合并筛选单抗。结果显示,经亚克隆纯化,共筛选获得12株阳性杂交瘤细胞株,即1B12、1E12、1H7、2B5、2C5、2C8、2D8、2F2、3C11、3D6、3G7和4G6。利用间接免疫荧光(IFA)、酶联免疫吸附法(ELISA)和免疫印迹检测单抗效价。成功制备了特异性鸡p21单抗。这为进一步研究鸡p21生物学功能奠定了基础。
To prepare monoclonal antibody against chicken p21,the p21 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) and subcloned into the prokaryotic expression vector,p ET-30 a.Therecombinant plasmid,designated as pET-30a-p21,was transformed into E.coli BL21(DE3)competent cells.Separated by SDS-PAGE,the induced protein was shown to be expressed as inclusionbodies.The positive bands were cut,ground and used as antigens to immunize BALB/c mice intraperitoneally for three times.The spleen was isolated and fused with SP2/0.Hybridoma clones that produced antibodies against p21 were screened by IFA,ELISA and limited dilution.The antibody levels were detected by indirect immunofluorescence assay(IFA),enzyme-linked immunosorbent assay(ELISA) and Western-blot.Twelve positive hybridoma clones were screened and identified as 1B12,1E12,1H7,2B5,2C5,C8,2D8,2F2,3C11,3D6,G7 and 4G6.The monoclonal antibody against p21 was prepared as a tool for the study of p21 functions.
To prepare monoclonal antibody against chicken p21,the p21 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) and subcloned into the prokaryotic expression vector,p ET-30 a.Therecombinant plasmid,designated as pET-30a-p21,was transformed into E.coli BL21(DE3)competent cells.Separated by SDS-PAGE,the induced protein was shown to be expressed as inclusionbodies.The positive bands were cut,ground and used as antigens to immunize BALB/c mice intraperitoneally for three times.The spleen was isolated and fused with SP2/0.Hybridoma clones that produced antibodies against p21 were screened by IFA,ELISA and limited dilution.The antibody levels were detected by indirect immunofluorescence assay(IFA),enzyme-linked immunosorbent assay(ELISA) and Western-blot.Twelve positive hybridoma clones were screened and identified as 1B12,1E12,1H7,2B5,2C5,C8,2D8,2F2,3C11,3D6,G7 and 4G6.The monoclonal antibody against p21 was prepared as a tool for the study of p21 functions.
引文
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[18]李圣爱.p21 WAF1/CIP1基因上游主要转录调控序列区及其相关功能[J].医学分子生物学杂志,2001,23(2):70-74.LI Sheng-ai.The transcriptional regulatory sequence and the function of p21 WAF1/CIP1 gene[J].Medical Molecular Biology Journal,2001,23(2):70-74.(in Chinese)
[19]李向荣,林星余,肖忠秀,等.p21结构功能与研究新进展[J].医学综述,2008,14(14):2094-2097.LI Xiang-rong,LIN Xing-yu,XIAO Zhong-xiu,et al.Progress of p21 about its structure,function and related research[J].Medical Recapitulate,2008,14(14):2094-2097.(in Chinese)
[2]JUNG Y S,QIAN Y,CHEN X.Examination of the expanding pathways for the regulation of p21 expression and activity[J].Cell Signal,2010,22(7):1003-1012.
[3]FANG F,JIAN P,LI Y P,et al.p21-activated kinase 1(PAK1)expression correlates with prognosis in solid tumors:A systematic review and meta-analysis[J].Oncotarget,2016,7(19):27422-27429.
[4]GARTEL A L,TYNER A L.The role of the cyclin-dependent kinase inhibitor p21 in apoptosis[J].Mol Cancer Ther,2002,1(8):639-649.
[5]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报:自然科学版,2008,5(3):77-82.XIE Ting-bo.The research progress of E.coli expression system[J].Journal of Yangtze University(Nat Sci Edit),2008,5(3):77-82.(in Chinese)
[6]高俊娜,张幸星,连雪,等.马立克病病毒Meq单克隆抗体的制备[J].中国兽医科学,2016,46(9):1073-1078.GAO Jun-na,ZHANG Xing-xing,LIAN Xue,et al.Generation of monoclonal antibodies against Marek’s disease virus oncoprotein Meq[J].Chinese Veterinary Science,2016,46(9):1073-1078.(in Chinese)
[7]付明娟,林接玉,谢捷明.包涵体蛋白复性的研究进展[J].医学综述,2015,21(20):3657-3659.FU Ming-juan,LIN Jie-yu,XIE Jie-ming.The research progress of inclusion body protein renaturation[J].Medical Recapitulate,2015,21(20):3657-3659.(in Chinese)
[8]姜静,孙其飞,陈勇,等.切胶免疫制备腮腺液高丰度蛋白多克隆抗体[J].北京口腔医学,2007,15(5):254-256.JIANG Jing,SUN Qi-fei,CHEN Yong,et al.Preparation and characterization of polyclonal antibodies against high abundant proteins in human parotid saliva[J].Beijing Journal of Stomatology,2007,15(5):254-256.(in Chinese)
[9]KARIMIAN A,AHMADI Y,YOUSEFI B.Multiple functions of p21in cell cycle,apoptosis and transcriptional regulation after DNA damage[J].DNA Repair(Amst),2016,42:63-71.
[10]宋其蔓,孙洞箫.细胞凋亡与p21关系的研究进展[J].医学综述,2008,14(6):816-818.SONG Qi-man,SUN Dong-xiao.Advancement in researches on relationship between apoptosis and p21[J].Medical Recapitulate,2008,14(6):816-818.(in Chinese)
[11]LIU S,BISHOP W R,LIU M.Differential effects of cell cycle regulatory protein p21(WAF1/Cip1)on apoptosis and sensitivity to cancer chemotherapy[J].Drug Resist Updat,2003,6(4):183-195.
[12]DATTO M B,WANG X F.Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism[J].Proc Natl Acad Sci USA,1995,92(12):5545-5549.
[13]ENGLERT C,MAHESWARAN S,GARVIN A J,et al.Induction of p21 by the Wilms'tumor suppressor gene WT1[J].Cancer Res,1997,57(8):1429-1434.
[14]SOMASUNDARAM K,ZHANG H,ZENG Y X,et al.Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDKinhibitor p21WAF1/Ci P1[J].Nature,1997,389(6647):187-190.
[15]KROKER A J,BRUNING J B.p21 exploits residue tyr151 as a tether for high-affinity PCNA binding[J].Biochemistry,2015,54(22):3483-3493.
[16]PRIVES C,GOTTIFREDI V.The p21 and PCNA partnership:a new twist for an old plot[J].Cell Cycle,2008,7(24):3840-3846.
[17]ZALDUA N,LLAVERO F,ARTASO A,et al.Rac1/p21-activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes[J].FEBS J,2016,283(4):647-661.
[18]李圣爱.p21 WAF1/CIP1基因上游主要转录调控序列区及其相关功能[J].医学分子生物学杂志,2001,23(2):70-74.LI Sheng-ai.The transcriptional regulatory sequence and the function of p21 WAF1/CIP1 gene[J].Medical Molecular Biology Journal,2001,23(2):70-74.(in Chinese)
[19]李向荣,林星余,肖忠秀,等.p21结构功能与研究新进展[J].医学综述,2008,14(14):2094-2097.LI Xiang-rong,LIN Xing-yu,XIAO Zhong-xiu,et al.Progress of p21 about its structure,function and related research[J].Medical Recapitulate,2008,14(14):2094-2097.(in Chinese)