Prokaryotic Expression Of PRGL And Preparation Of Its Antibody

PRGL is a new gene isolated from Gerbera petals in our lab. It contains ahomological domain of GASA and a similar structure of PRP. It is presumed thatPRGL might be involved in GA induced growth and localized in the cell wall. In thepresent study, polyclonal antibodies were prepared by constructing of recombinantprokaryotic expression plasmid in order to study the functions of PRGL. The mainresults are as follows:The plasmid pET32a was used to construct the recombinant prokaryoticexpression plasmid pET32a-PRGL. After DNA sequencing identification, the E. coliwas induced under the conditions of bacteria initial concentration OD₆₀₀=0.6, IPTGconcentration was 0.6 mM and inducing time was 6h at 28℃. The target proteinpeptide obtained was 34.76kD. The pET30c was used to construct plasmidpET30c-PRP followed by the same procedure as pET32a-PRGL production exceptIPTG concentration was at 1.0 mM. The target protein peptide obtained was 13.64 kD.His marker was used to check the target proteins in our experiment.After quantification, the prokaryotic expressed proteins were injected into rabbitsto raise anti-PRGL and anti-PRP antibodies. The serum was collected from auricularvein of rabbits and stored under -80℃as control. Polyclonal antibodies were checkedby indirect ELISA after three-time immunization. The results showed that they werestill immunize-active after diluted 600×.Proteins from E. coli induced by IPTG were used as antigens, immune andnon-immune serums were used as antibodies. The validity of PRGL protein andpolyclonal antibodies were confirmed by Western Blot.Total proteins were extracted from P2 and P6 petals of Gerbera and separated ona SDS-PAGE, transferred to a nylon membrane. Western blot was performed usingantibody against Gerbera PRGL. A specific band was found in P2 petals. The reasonof the appearance of this band was discussed.