Prokaryotic expression of human mitochondrial transcription termination factor 3 and preparation of its polyclonal antibody
10.16571/j.cnki.1008-8199.2016.10.003
- VernacularTitle:人MTERF3蛋白的原核表达与多克隆抗体制备
- Author:
Yongqin YANG
;
Chenggui ZHANG
;
Meitao SUN
;
Yun WANG
;
Zefang YANG
;
Xiaojuan ZHANG
;
Wei XIONG
- Publication Type:Journal Article
- Keywords:
Human mitochondrial transcription termination factor;
Prokaryotic expression;
Purification;
Polyclonal antibody
- From:
Journal of Medical Postgraduates
2016;29(10):1020-1025
- CountryChina
- Language:Chinese
-
Abstract:
Objective Human mitochondrial transcription termination factor 3 ( MTERF3 ) is a negative regulator of mito?chondrial gene expression and energy metabolism. This study was to construct a prokaryotic expression system for MTERF3 in Esche?richia coli ( E. coli ) and prepare its mouse?anti?human polyclonal antibody. Methods The complete open reading frame ( ORF) of human MTERF3 cDNA was amplified by RT?PCR and subcloned into prokaryotic expression vector pET28b. Then the recombinant plasmid pET28b?MTERF3 was transformed into competent E.coli BL21(DE3) and IPTG induced the expression of 6×his fusion protein. The recom?binant human MTERF3 protein was purified through Ni2+?NTA agar?ose gel column affinity chromatography and the purified recombinant protein was used as immunogen to immunize the BALB/c mice to pre?pare its specific polyclonal antibody. The titer and specificity of the antibody were analyzed by ELISA, Western blot and cellular immuno?fluorescence, respectively. Results The recombinant human MTERF3 protein was successfully expressed in E. coil and the mouse?anti?human MTERF3 polyclonal antibody with high quality was successfully prepared. ELISA showed that the titer of the antibody was 1:105 . Western blot and immunofluorescence detection revealed that the mouse?anti?human MTERF3 antibody could recognize the native MTERF3 antigen specifically. Conclusion Human MTERF3 expressed in the prokaryotic system has strong immunogenicity and the polyclonal antibody obtained from immunizing mice has high titer and specificity. The prokaryotic expression of human MTERF3 and the preparation of its antibody lay the foundation for further function research of human MTERF3.
