| Antimicrobial peptides (AMPs) are short cationic peptides with an amphiphilic nature. They play an important role in the innate immunity of host organisms, including animals, plants, and humans. AMP which with high effective and broad-spectrum antibacterial activities and hardly inducing drug-resistance. The AMPs are promising candidates for novel anti-infective drugs because they are effective against antibiotic-resistant bacteria.Extracting nature antimicrobial peptides have the limits of little raw material,high cost, and complex process, it has great economic value to use of genetic engineering technology produce high activity antibacterial.1 In this study, three primers which have complementary areas were designed and synthesized according to the Dermadistinctin-L gene sequence reported in GenBank. and obtained the whole gene by PCR method, primers which containing Sac I and HindⅢrestriction enzyme sites was designed,and enterokinase signal cleavage site was introduced into the two ends of Dermadistinctin-L gene.2 The amplicon was inserted into a bacterial plasmid pET-32a(+) vector, and identification by restriction enzyme digestion and sequencing to the Purpos,then transfected into E. coli BL21 (DE3).after induced by IPTG and detected by SDS-PAGE, analysis showed that Fusion Dermadistinctin-L Protein was sueeessfully expressed.3 As temperature,IPTG concentration and induction time were optimized,the recombinant protein was high expressed at 37℃for 5h with 0.1M IPTG induction. the expression of the subunit was detected, and it could account for 30% of the total host protein。The expressed protein was one-step purified to 80% using Ni-IDA affinity chromatography method under the optimum condition.4 Fusion protein was eleaved by enterokinase and identification of its activity, the study of antibacterial activity in vitro exhibit that it has doughty antibacterial activity to gram negative and gram positive bacterium.
|
PDF Full Text Request