Construction Of PET-28b-TatPTD-hbFGF, Purification Of Expression Product, Penetration Of Recombinant Protein On Cell Membrane

Objective To construct the recombinant vector which can express TatPTD-hbFGF in E.Coli, express and purify the target protein in vitro and detect biological activity of recombinant hbFGF. To study whether or not is the recombinant protein to penetrate into cell membrane. Methods PCR was used to amplify TatPTD-hbFGFh DNA, and the PCR product was ligated into the pGEM-T Vector to get the recombinant plasmid pGEM-T-TatPTD-hbFGFh. After proved by restrictive endonuclease mapping, the recombinant plasmid was amplified, digested by the restriction endonuclease (EcoRI and Nde I). And the target gene, TatPTD-bFGF, was inserted into pET-28b to construct expression vector. The positive clones were screened by blue-white color screen and antibiotic resistance, and were proved by restrictive endonuclease mapping analysis and DNA sequencing. IPTG was used to induce the TatPTD-bFGF expression. The target protein was purified by anion exchang column and detected biological activity of recombinant hbFGF. We checked the expression of TatPTD-bFGF in endothelial cell by immunochemistry and Westen blotting. Results the constructed plasmid was demonstrated by restriction endonucleases mapping analysis and DNA sequencing. SDS-PAGE showed that the expressed products was about 21.5kD and the quantity was about 24% of the whole protein of the bacteria. After examining the cells by immunoftuorescent and Westen blotting, we find that TatPTD can be mediately deliver biologically active protein into cell. Conclusion The expressed plasmid was constructed. TatPTD-hbFGF not only was expressed in E. Coli, and purify the fusion protein have biological activity, but also TatPTD was identified that have mediate bigger protein to pass through cell membrane.