Bioinformatic investigation of plant beta-defensin peptide and cloning of this peptide with pAMJ1653 vector in Lactococcus lactis

Frequent and excessive use of antibiotics has caused the resistance of pathogenic bacteria to common antibiotics. Considering this issue, researchers are looking for new methods to replace antibiotics, which can be referred to as antimicrobial peptides (AMP). Therefore, the aim of the present study was bioinformatic investigation of the effect of his taq addition on the antibacterial activity of plant beta-defensin peptide and clone this peptide gene in the expression vector pAMJ1653 in Lactococcus lactis bacteria. The third structure of beta defensin peptide with his-taq was predicted through I-TASSER and verified by SAVE 6 and prosA software. Finally, molecular docking was performed through Cluspro software between beta defensin with /without his-taq sequence and Escherichia coli LPTD antigen. In the exprimental section, beta defensin gene was used along with his-taq sequence and secretion signal in pGEM-DFF replication vector and DH5α replication host. After plasmid extraction and enzymatic digestion by Sap1 and Sal1 enzymes, plant beta defensin gene was homogenized into pAMJ1653 expression vector and transferred into Lactococcus lactis strain 1543 bacteria using electroporation method. The positive colonies containing the recombinant vector were checked by PCR colony method and finally enzymatic digestion was performed. In the bioinformatics part, the third structure of beta defensin was predicted with acceptable accuracy. Docking results showed that beta defensin peptide with/without his-taq is in contact with LPTD antigen in the right position. In the laboratory section, it was shown that the gene encoding plant betadefensin was successfully cloned in the pAMJ1653 expression vector