Recombinant Expression And Antimicrobial Activity Of Rat β-defensin-2

The infectious disease is extremely important topic in the medical study from ancient times till now. Although the antibiotic has played a tremendous role in the preventing and controlling microorganism infection, but because of the long-term abuse of antibiotics, the bacterium drug resistance question already became the puzzle medical problem. Overcoming the problem of bacterial resistance becomes the urgent matter. Therefore, it has very important practical significance to find and develop non-drug resistance new antibacterial drugs. Antimicrobial peptides are an important part as a biological machine body's natural immune system. It has anti-bacterial, anti-virus, anti-fungal, anti-tumor and other biological activity. Because they unique resist bacteria mechanism as well as the pathogenic microorganism not easy to have the drug resistance, it has a potential application as a new antibiotic.Objective:Construction a eukaryotic expression vector of recombinant rat P-defensin-2 (rBD2), which was transformed into the Mouse fibroblast(L929) for expression. Then, detect the expression of target gene and antibacterial activity of purpose protein. In order to solve the problem of bacterial resistance to provide theoretical and experimental basis.Method:The total RNA was extracted from rat lung tissue as template, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the full length of coding region of rBD2 gene. Then we constructed a eukaryotic expression plasmid pCAGG-rBD2, and the plasmid was transfected into L929 by Lipofection. The expression of rBD2 in L929 cells was verified by RT-PCR and western blotting. Additionally, Using K-B disk diffusion method to detect biological activity of expression products.Results:RT-PCR amplified fragment about 230bp, connected to the cloning vector. The cloning vector was correct by DNA sequencing, it was correct that constructon of recombinant enkaryotic expression vector pCAGG-rBD2 by DNA sequencing, and the ligation was correct, too. The rBD2 gene was expressed in L929 cell which was transfected eukaryotic expression plasmid pCAGG-rBD2. Importantly, the cells can secrete biologically active rBD2.Conclusion:RBD2 gene was successfully cloned, and successfully constructed the eukaryotic expression vector pCAGG-rBD2. Finally, rBD2 gene can be effectively expressed in L929 cell, and the expression products have biological activity of killing Staphylococcus aureus.