Preparation Of The Breast Cancer Transductive Peptide And EGFP Fusion Protein And Its Intercellular Transduction Activity In MDA-MB-231

Objective To construct combinated prokaryotic vector pET-28a (+)-PI-EGFP contained reast cancer specific transductive peptide(PI) and enhanced green fluorescent protein(EGFP),purified fusion protein of breast cancer-specific transduction peptide PI with EGFP,certificate its transduction activity in MDA-MB-231, Preparation of mouse model of breast cancer; inject via the tail vein and in tumor to observate its distribution in tumor-bearing nude, it made an expeiment foundation for PI as a targeting vehicle for in vivo studies in future.Methods First synthesis DNA sequence of PI, insert it into eukaryotic expression vector pEGFPN2, digested by enyzme to obtain fusion fragment PI-EGFP, then insert it into the prokaryotic vector pET-28a(+)to get pET-28a(+)-PI-EGFP. After induced by IPTG at final concentration lmmol/L, Purify the protein use its N-terminal His-tag.The specific protein expression was detected by SDS-PAGE and Western Blotting. Fluorescence microscope was used to detect EGFP activity of the fusion protein. Detect conduction ablitity of the specific fusion protein by co-cultured it with breast cancer cell MDA-MB-231; inject the fusion protein via tail vien and local tumor position,observate its distribution in vivo.Results After sequencing identification, pET-28a (+)-PI-EGFP was constructed successfully. The fusion protein was expressed effectively after induction with IPTG. Besides, it is soluble. The specific fusion protein had an apparent related molecular weight about 33 kD as indicated by SDS-PAGE analysis, which in line with the theoretical value. And it was identified by Western Blotting.The fusion protein was observed to emit green fluorescence at provocate wave 490nm.By co-cultrue with breast cancer MDA-MB-231, green fluorescence could detected in the cells; green fluorescence can not observed Via the tail vein after injection of fusion protein in various tissues and organs, but green fluorescence can be seen via inject at the tumor position obviously.Conclusion The prokaryotic vector pET-28a (+)-PI-EGFP can effectively express PI-EGFP fusion protein, and show stabile transductive character, PI could transduct EGFP to the local tumor position,laying a foundation for further study of the transduction fuction about PI and further research on its transmembrane conductance mechanism.