[Preparation and identification of monoclonal antibodies against chicken cell cycle checkpoint kinase 2 (cChk2)].
Objective To prepare monoclonal antibodies (mAbs) 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 pGEX-4T-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 1F4, 2D9, 2G1, 3D9, 3E3, 4B5, 4E2, 5C9 and 5F7. Conclusion The study has prepared mAbs against cChk2 with a good specificity and a high titer.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Junna
J
Gao
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Xue
X
Lian
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Haiwei
H
Sun
Shanghai Veterinary Research Institute, China Academy of Agricultural Sciences, Shanghai 200241, China.
Zejun
Z
Lu
Anhui Key Laboratory of Poultry Infectious Disease Prevention and Control, Anhui Science and Technology University, Fengyang 233100, China.
Xunhai
X
Zhang
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Yong-Sam
YS
Jung
Shanghai Veterinary Research Institute, China Academy of Agricultural Sciences, Shanghai 200241, China. *Corresponding authors, E-mail: yqian@njau.edu.cn.
Hongjun
H
Chen
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. *Corresponding authors, E-mail: vetchj@shvri.ac.cn.
Yingjuan
Y
Qian
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000276:immunology*; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D064447:Checkpoint Kinase 2