Yong Liu
Southern Medical University, China
Title: Construction of PEP-CCL6 fusion protein expression plasmids for 293 cell lines
Biography
Biography: Yong Liu
Abstract
Background: The increasing spread of antibiotic-resistant microorganisms has led to the need of developing alternative antimicrobial treatments. CCL-6 has shown antibacterial activity in vitro. However, due to the poor permeability and selectivity of the cell membrane, the effect on bacteria in vivo is not ideal. The use of CCL-6 fused with PEP, a cell-penetrating peptide is a promising approach to enhance the antibacterial ability of CCL-6.
Methods: The His-CCL6-PEP1 coding sequence was amplified by polymerase chain reaction (PCR) and then cloned into the eukaryotic expression vector (pABP) containing His tag. The constructed vector, verified by restriction endonuclease digestion, PCR and DNA sequencing, was then transformed into HEK 293 cell for expression. The expression of His-CCL6-PEP1 recombinant protein was purified with Ni2+-NTA affinity chromatography method and identified by SDS-PAGE and Western blotting.
Results: PEP-CCL6 was produced from 293 cell lines. The successful construction of recombinant plasmid was confirmed by restriction digestion, PCR and sequencing. The molecular weight of the purified fusion protein was identified as 15kD by SDS-PAGE, which was identical to the expected value. It was confirmed by western blotting that is CCL6-PEP1 fusion protein could be recognized by His monoclonal antibody.
Conclusions: The recombinant plasmid of pABP-CCL6-PEP1 has been successfully constructed. The expressed His- CCL6-PEP1 fusion protein has been purified and identified