Prokaryotic Expression And Preparation Of Polyclonal Antibodies Of PwHAP5and PwFKBP12in Picea Wilsonii

Picea wilsonii as a unique specie of our country has strong ability to resist cold and it is a typical gymnosperms. HAP, a kind of important transcription factor, occurs in yeasts, mammals and plants. It can bind to the CCAAT box and regulate the expression of target genes. HAP complex includes three subunits. HAPS, which is one of the subunits has a very important significance to drought stress. FKBPs are a highly conserved partner molecule protein family. It can combine with immune inhibitors like rapamycin FKBP12gene in plant which may play the role of regulation under adversity stress. In this research, polyclonal antibodies against two genes were made to analysis localization of protein in the pollen tube of Picea wilsonii.The cDNA encoding full-length of FKBP12and HAP5were amplified by PCR and were inserted into vector pET-48b. The recombinant plasmids were transformed into E.coli BL21then and the recombinant protein of33kD and45kD was expressed by induction with IPTG. The expression product was identified by SDS-PAGE and Western blotting assays. The fusion proteins were purified by affinity chromatography and then immunized rabbits to generate polyclonal antibody. The polyclonal antibodies can be used for indirect immunofluorescence to analysis localization of protein in the pollen tube. The obtained results are as follows:1. The cDNA encoding full-length of FKBP12and HAP5were identified by agarose gel electrophoresis and sequencing.2. Colonies PCR and sequencing proved that the prokaryotic expression vector with target gene was constructed successfully and a large number of target protein expression was obtained.3. High purity of purpose protein was attained by affinity chromatography and then immunized it to rabbits to generate polyclonal antibody. 4. Successfully acquired the polyclonal antibody, preparation of FKBP12and HAP5rabbit antiserum titer in1:729000, ELISA results showed that the fusion protein with good immunogenicity. Indirect ELISA detection antibody titer purification, show that after purification of FKBP protein and HAP5protein of rabbit polyclonal antibody titer are very high, detection sensitivity for16ng/mL. The polyclonal antibodies could be used for further investigation, provided a theoretical basis for further research.5. The pollen tube deal with Polyclonal antibody and FITC AffiniPure Goat Anti-Rabbit IgG(H+L). Indirect immunofluorescence established the foundation of FKBP12and HAP5gene in protein level.