Construction Of PGEX-6P-1-opn, Purification Of Expression Product, Preparation Of Polycolonal Antibody And Its Preliminary Application

Objective To construct the recombinant vector pGEX-6P-l-opn, induce expression of the recombinant protein in XL1-blue, purify and cleave fusion protein. To prepare rabbit anti-hOPN polycolonal antibody, separate and purify IgG, coupling IgG and HRP, establish Sandwich ELISA methods. Methods To culture HUVEC and prepare total RNA, synthesize cDNA first chain by reverse transcription(RT). PCR was used to amplify opn fragment , which was inserted into pGEX-6P-l to construct expression vector. To transform the recombinant vector into E.coli XL1-blue, induce the expression by adding IPTG, centrifuge and resuspend pellet, lyse the cells using a appropriate sonicator. To purify the fusion protein on Glutathione Sepharose 4B affinity chromatograph column , cleave it using PreScission Protease. To select healthy Newzealand rabbit, inject recombinant rhOPN many times at different points subcutaneously, check titration using indirect ELISA. When titration exceed 1:160, the blood was collected from aorta. IgG was Separated, purified with CM-SepHarose FF and SAS and labelled with HRP using improved NaIO4 method. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid is the recombinant pGEX-6P-l-opn. A new band of protein was found in SDS-PAGE. The fusion protein was eluted from affinity column that is about 85Kd, 58Kd rhOPN and 26Kd GST are obtained after cleavage. Acquire 1:6400 polycolonal antibody, purify IgG using CM-Sepharose FF and SAS. Contusion To construct and induce expression of pGEX-6P-1-opn recombinant, purify fusion protein and cleave it. To prepare and purify IgG, label it with HRP and establish Sandwich-ELISA method successfully.