The Establishment Of Efficient Expression System Of Antibacterial Peptide PNK-19in Bacillus Subtilis

Antimicrobial peptides is widely recognized as the best substitute for thetraditional antibiotics, with small molecular weight, better thermal stability, betterwater solubility, broad spectrum of activity against microorganism. But they are alsorestrictions on the source of the antimicrobial peptides. Bacillus subtilis expressionsystem can achieve high throughput expression of antimicrobial peptides and take theadvantage of the nature of Bacillus subtilis to overcome the question of E. coliexpression system. Moreover, the molecular genetic background of Bacillus subtilis isclear. It breed fast, the culture condition is simple. These character of Bacillus subtilissuitable for large-scale industrial production providing solutions for the source ofantimicrobial peptides.Antimicrobial peptides are usually constitute of10to30amino acids. And titsspatial structure is not complicated, so the more research also are needed on theseantimicrobial peptides. In this experiment, the two short peptides are chosen from theNCBI and added different restriction sites at its C-terminus, then named as PNK-19、LNK-16and LNF-14. These peptides analogs are synthesized and test theantibacterial activity of those peptides. The result shows that PNK-19express highantibacterial activity against Gram-positive bacteria, Gram-negative bacteria andfungi.In order to increase the expression of target protein, this reaserch choose the wayof series expression. Three tandem genes of PNK-19are synthesized and named asPNK-19-3E, which with the restriction enzyme cut site of EcoR Ⅰand Xbal Ⅰ at theupstream and downstream. The pHT304expression vector and the target gene aredigested with the restriction enzyme of EcoR Ⅰand Xbal Ⅰ. Target gene andexpression vector were connected by T4DNA ligase, which was named of thepHT304-PNK-19-3E. The recombinant plasmid was tested by restriction enzymedigestion, PCR and check the sequence of the gene of expression vector.In order to obtain high activity antimicrobial peptides. Bacillus subtilis WB800 was chosen as the host cell, which is knocked out8genes of endocrine protein. Therecombinant plasmids pHT304-PNK-19-3E was transformed into competent cells.The result of Tricine-SDS-PAGE shows that the target protein is about13.5kD in thesupernatant. The protein is purified by SDS-PAGE and digested by endopeptidaseenzyme-K. Then the antibacterial activity was tested. The result shows that the targetprotein after digested have a obvious activity against bacteria.