Construction Of Expression Vector Of Rat Beta-defensin-2 Gene And Expression In Escherichia Coli

BackgroundBeta defensins are small cationic peptides. Beside the antimicrobial function, beta-defensins have multiple roles in innative immunity and acquired immunity. Beta-defensin-2 is inducible expressed throughout the epithelium and the serous gland cells in the airway walls. The deficiency of hBD-2 would cause CF (Cystic fibrosis of the lung) and infection. Antimicrobial peptides qualify as prototypes of innovative drugs that might be used as antibiotics or anti-tumor modifiers.It is difficult to achieve amount of antimicrobial peptides from nature sources. Genetic engineering has been utilized to produce small cationic peptides for a decade. But the techniques for beta-defensin-2 is not consummate. E. coli is the most used host cell in genetic engineering with a fastgrowth rate and well established expression systems. However, there are two major barriers in using E. coli as the host for rBD-2 expression which include the anti-Gram-negative bacterial activity of rBD-2, and the susceptibility of the small peptide to proteolytic degradation. Such difficulties may be avoided by using a carefully designed plasmid to prevent toxic rBD-2 activity, and by selecting an E. coli strain with low protease activity. Furthermore, cell growth and product expression should be divided into different phases during the cell culture.ObjectiveBeta-defensin-2 is a important mediator in either innate immunity or anti-infection. It is difficult to achieve amount of antimicrobial peptides from nature sources. The object of this study is to construct the expression vector of rat beta-defensin-2 gene and study the expression of the recombinant expression in E. Coli.Materials and MethodsThe rBD-2 DNA fragment was amplified by RT-PCR from the total cellular RNA of the lung tissue. The pET-32a(+) vectorwas cleaved by the restriction enzymes Ncol I and HindIII, the larger fragment was recovered. The rBD-2 DNA fragment was digested by the restriction enzymes Ncol I and Hindlll (the restriction sites were introduced through PCR primers). The expression vector, pET32~rBD2, was constructed through a ligation reaction. A fresh clone of E. coli , harboring the pET32~rBD2vector, was grown in MBL medium . When the cells have been cultured to logarithm growth period, expression was induced with 0. 8mM isopropylthio- æ‹¢ -D-galactoside (IPTG).The cells were harvested. SDS-PAGE and western blotting were done to analysis the result.Result1. A 152bp DNA fragment was successfully amplified by RT-PCR from the total cellular RNA;2. The expression vector pET32-rBD2 was well constructed;3. The target fusion protein was expressed in E. coli BL21 (DE3).ConclusionIn this study, an antimicrobial peptide, rBD-2 was high-level expressed in E. coli as a fusion protein. However, further work should be done to establish an effectivepurification process.