[Prokaryotic expression of F10 and preparation of its polyclonal antibody].
OBJECTIVE
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.


CONCLUSIONS
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.
Fu
Xin
X
Experimental Medical Research Center, Guangzhou Medical College, Guangzhou 510182, China. fuxin@gzhmc.edu.cn
Zou
Dong-ting
DT
Zhou
Wen-qu
WQ
Xing
Fu-qi
FQ
Li
Bing
B
D000906:Antibodies / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D012995:Solubility