鸡细胞周期检验点激酶2(cChk2)单克隆抗体制备和鉴定
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摘要
目的制备特异性鸡细胞周期检验点激酶2(cChk2)单克隆抗体(m Ab)。方法反转录PCR扩增cChk2基因,将其克隆入原核表达质粒p GEX-4T-3中,在BL21(DE3)中经异丙基硫代半乳糖苷(IPTG)诱导表达,经SDS-PAGE检测可溶性。用cChk2腹腔免疫BALB/c小鼠,利用间接免疫荧光法(IFA)和Western blot法检测抗血清;血清检测阳性后,取小鼠脾脏与小鼠骨髓瘤细胞Sp2/0融合,利用IFA和有限稀释法筛选阳性单克隆杂交瘤细胞株。结果 cChk2主要以包涵体形式存在,多抗血清具有良好的效价,获得9株阳性杂交瘤细胞株,即1F4、2D9、2G1、3D9、3E3、4B5、4E2、5C9及5F7。结论成功制备了特异性良好的cChk2 m Ab。
Objective To prepare monoclonal antibodies( m Abs) against chicken cell cycle checkpoint kinase 2(cChk2). Methods The cChk2 gene was amplified by reverse transcription PCR(RT-PCR) and subcloned into the prokaryotic expression vector p GEX-4 T-3. After induced by IPTG,cChk2 was expressed in BL21( DE3) E. coli cells and analyzed by SDS-PAGE to determine its soluability. BALB/c mice were immunized with cChk2 protein peritoneally. Indirect immunofluorescence assay( IFA) and Western blotting were used to detect anti-serum; if the detection result was positive,IFA and limited dilution was performed to screen hybridoma clones that produced antibodies against cChk2. Results cChk2 was mainly expressed in inclusion bodies. The anti-sera were able to recognize Chk2. Nine positive hybridoma clones were obtained and identified as 1 F4,2 D9,2 G1,3 D9,3 E3,4 B5,4 E2,5 C9 and 5 F7. Conclusion The study has prepared m Abs against cChk2 with a good specificity and a high titer.
Objective To prepare monoclonal antibodies( m Abs) against chicken cell cycle checkpoint kinase 2(cChk2). Methods The cChk2 gene was amplified by reverse transcription PCR(RT-PCR) and subcloned into the prokaryotic expression vector p GEX-4 T-3. After induced by IPTG,cChk2 was expressed in BL21( DE3) E. coli cells and analyzed by SDS-PAGE to determine its soluability. BALB/c mice were immunized with cChk2 protein peritoneally. Indirect immunofluorescence assay( IFA) and Western blotting were used to detect anti-serum; if the detection result was positive,IFA and limited dilution was performed to screen hybridoma clones that produced antibodies against cChk2. Results cChk2 was mainly expressed in inclusion bodies. The anti-sera were able to recognize Chk2. Nine positive hybridoma clones were obtained and identified as 1 F4,2 D9,2 G1,3 D9,3 E3,4 B5,4 E2,5 C9 and 5 F7. Conclusion The study has prepared m Abs against cChk2 with a good specificity and a high titer.
引文
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[15]Zachos G,Rainey M D,Gillespie D A.Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects[J].EMBO J,2003,22(3):713-723.
[16]Rainey M D,Black E J,Zachos G,et al.Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase[J].Oncogene,2008,27(7):896-906.
[17]Zachos G,Rainey M D,Gillespie D A.Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures[J].Mol Cell Biol,2005,25(2):563-574.
[18]Churikov D,Price C M.Pot1 and cell cycle progression cooperate in telomere length regulation[J].Nat Struct Mol Biol,2007,15(1):79-84.
[19]Mocciaro A,Berdougo E,Zeng K,et al.Vertebrate cells genetically deficient for Cdc14 A or Cdc14 B retain DNA damage checkpoint proficiency but are impaired in DNA repair[J].J Cell Biol,2010,189(4):631-639.
[20]Ta H Q,lvey M L,Frierson H F,et al.Checkpoint kinase 2 negatively regulates androgen sensitivity and prostate cancer cell growth[J].Cancer Res,2015,75(23):5093-5105.
[21]Nuc P,Nuc K.Recombinant protein production in Escherichia coli[J].Postepy Biochem,2006,52(4):448-456.
[22]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.
[23]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.
[24]季林,刘佩娟,王毅,等.GST融合蛋白包涵体纯化方法的探索[J].科学技术与工程,2012,12(5):1014-1016.
[25]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.
[2]Turnell A S,Grand R J.DNA viruses and the cellular DNA-damage response[J].J Gen Virol,2012,93(Pt 10):2076-2097.
[3]Stolz A,Ertych N,Bastians H.Tumor suppressor CHK2:regulator of DNA damage response and mediator of chromosomal stability[J].Clin Cancer Res,2011,17(3):401-405.
[4]Ahn J Y,Schwarz J K,Piwnica-Worms H,et al.Threonine 68phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation[J].Cancer Res,2000,60(21):5934-5936.
[5]Xu X,Tsvetkov L M,Stern D F.Chk2 activation and phosphorylationdependent oligomerization[J].Mol Cell Biol,2002,22(12):4419-4432.
[6]Chou W C,Hu L Y,Hsiung C N,et al.Initiation of the ATM-Chk2DNA damage response through the base excision repair pathway[J].Carcinogenesis,2015,36(8):832-840.
[7]Kim S,Lee H S,Ji J H,et al.Hepatitis B virus X protein activates the ATM-Chk2 pathway and delays cell cycle progression[J].J Gen Virol,2015,96(8):2242-2251.
[8]Sur S,Agrawal D K.Phosphatases and kinases regulating CDC25activity in the cell cycle:clinical implications of CDC25 overexpression and potential treatment strategies[J].Mol Cell Biochem,2016,416(1/2):33-46.
[9]Ertych N,Stolz A,Valerius O,et al.CHK2-BRCA1 tumor-suppressor axis restrains oncogenic Aurora-A kinase to ensure proper mitotic microtubule assembly[J].Proc Natl Acad Sci U S A,2016,113(7):1817-1822.
[10]Parameswaran B,Chiang H C,Lu Y,et al.Damage-induced BRCA1phosphorylation by Chk2 contributes to the timing of end resection[J].Cell Cycle,2015,14(3):437-448.
[11]Hazar-Rethinam M,Endo-Munoz L,Gannon O,et al.The role of the E2 F transcription factor family in UV-induced apoptosis[J].Int J Mol Sci,2011,12(12):8947-8960.
[12]Nikitin P A,Luftig M A.The DNA damage response in viral-induced cellular transformation[J].Br J Cancer,2012,106(3):429-435.
[13]Ciccia A,Elledge S J.The DNA damage response:making it safe to play with knives[J].Mol Cell,2010,40(2):179-204.
[14]Liu Y,Li Y,Lu X.Regulators in the DNA damage response[J].Arch Biochem Biophys,2016,594:18-25.
[15]Zachos G,Rainey M D,Gillespie D A.Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects[J].EMBO J,2003,22(3):713-723.
[16]Rainey M D,Black E J,Zachos G,et al.Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase[J].Oncogene,2008,27(7):896-906.
[17]Zachos G,Rainey M D,Gillespie D A.Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures[J].Mol Cell Biol,2005,25(2):563-574.
[18]Churikov D,Price C M.Pot1 and cell cycle progression cooperate in telomere length regulation[J].Nat Struct Mol Biol,2007,15(1):79-84.
[19]Mocciaro A,Berdougo E,Zeng K,et al.Vertebrate cells genetically deficient for Cdc14 A or Cdc14 B retain DNA damage checkpoint proficiency but are impaired in DNA repair[J].J Cell Biol,2010,189(4):631-639.
[20]Ta H Q,lvey M L,Frierson H F,et al.Checkpoint kinase 2 negatively regulates androgen sensitivity and prostate cancer cell growth[J].Cancer Res,2015,75(23):5093-5105.
[21]Nuc P,Nuc K.Recombinant protein production in Escherichia coli[J].Postepy Biochem,2006,52(4):448-456.
[22]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.
[23]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.
[24]季林,刘佩娟,王毅,等.GST融合蛋白包涵体纯化方法的探索[J].科学技术与工程,2012,12(5):1014-1016.
[25]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.