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Published on: January 17, 2015
[Prokaryotic expression of F10 and preparation of its polyclonal antibody]
Xin Fu1, Dong-ting Zou, Wen-qu Zhou
1Experimental Medical Research Center, Guangzhou Medical College, Guangzhou 510182, China. fuxin@gzhmc.edu.cn
Aim:
To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it.
Methods:
The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA.
Results:
Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000.
Conclusion:
F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.
