The Establishment Of Efficient Expression System Of PNK-19Antibacterial Peptide In Lactobacillus Acidophilus

Because of the extensive use of antibiotics, the frequent emergence of resistantbacteria has resulted in a significant threat to human health. With the properties of smallmolecular weight, no allergic reactions and unique mechanism of antibacterial,Antimicrobial peptides (AMPs) have been investigated as an efficient candidate for thetraditional antibiotics. Many naturally AMPs have a wide antimicrobial spectrum againstbacteria, fungi, parasites, viruses and tumor. AMPs are a part of the innate immune systemof many animals and plants. They protect the host organism against bacteria and fungalattacks by destroying the barrier function of the invading microbe’s membrane. However,the acquisition of the antimicrobial peptides problem has always been a bottleneck, whichrestricts the research and development of AMPs. To solve this problem, three series of genesof antibacterial peptides was expressed in probiotics. As natural AMPs could kill manyengineered bacteria, this method could make AMPs lose effectiveness provisionally. Thisresearch lays a foundation for the development and application of antimicrobial peptides.Protegrins-1and Indolicidin were chosen through bioinformatics method for theirsmall molecular weight and good antibacterial activity. After the analysis of acidity orbasicity, hydrophily and hydrophobicity of the peptides chain, endopeptidase-K,chymotrypsin-F and kallikrein-PRF were respectively added to the C terminus ofantibacterial peptides to form derivatives of AMPs(PNK-19, LNF-14and LNK-16). ThePNK-19which is made of Protegrins-1and endopeptidase-K was chosen. The protein ofPNK-19was translated into gene, XbalⅠand HindⅢ were added to the ends of the threeseries of genes.The target gene was228bp. The pMG36e and target genes were doubleenzymed respectively by restriction enzymes Xbal Ⅰ and Hind Ⅲ, and connected by T4DNA ligase. The recombinant plasmid is named of pMG36e-PNK-19-3E. The recombinantplasmid is tested by restriction enzyme digestion, PCR and check the sequence of the geneof expression vector The results showed that the expression vector pMG36e-PNK-19-3E ofLactobacillust was successfully constructed.pMG36e-PNK-19-3E was electrotransformed into the lactobacillus acidophiluscompetent cell. The results of SDS-PAGE and Western blot showed that the target proteinwas about10kD after expression. The protein was purified by SDS-PAGE and digested byendopeptidase enzyme-K. The antibacterial activity was tested by agar plate diffusionmethod. The result shows that the target protein possess activity against E. coli and S. aureus.