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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Published on: September 28, 2017
[Expression and biological function of ULBP4 in Rosetta-gamiTMB(DE3) and mAb preparation]
We Cao1, Zhi-Yong Hao, Xue-Yan Xi
1Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China. caowei_111@yahoo.com.cn
Aim:
To express human ULBP4 in Rosetta-gami(TM) B(DE3) and to prepare monoclonal antibody against ULBP4 for the research of gamma deltaT cells recognition mechanism.
Methods:
DNA fragments of ULBP4 were derived from HO-8910 RNA by reverse-transcriptase polymerase chain reaction(RT-PCR). The fragments encoding the former 225 amino acids of ULBP4 were cloned into Histag fusion protein expression vector pET22b(+). The C-Histag fusion ULBP4(225a) protein was expressed in inclusion body and purified step by step according to manufactory's protocol and renatured in bag filter. It's functional effect on NK cells was evaluated by NKG2D binding assay and IFN-gamma secretion experiments. Prokaryotic expressed human ULBP4 was used as an antigen to prepare monoclonal antibodies by means of the B lymphocyte hybridoma technique.
Results:
ULBP4 recombinant protein can stimulate NK cells to secrete IFN-gamma. Through PEG fusion and screening by limited dilution, we obtained four strains of hybridoma cells secreting anti-ULBP4 antibodies.
Conclusion:
The fusion protein was expressed successfully and functional. At the same time, the anti-ULBP4 mAbs were prepared successfully. Both of them provide a platform for further research.
