Abstract:

Objective The human-derived antimicrobial peptide（AMPs） histatin 5 （Hst5） and LL-37 were used as the parents to design a novel heterozygous peptide H5LL， and to construct a recombinant lactic acid bacteria expression vector by genetic engineering technology to achieve the efficient and safe expression of the AMPs. Methods Bioinformatics method was used to predict the physicochemical parameters， hydrophilicity/hydrophobicity， shear sites， phosphorylation sites， signal peptides and transmembrane regions， subcellular localization， and secondary structure of H5LL； the target sequence and the empty plasmid pMG36e were doubly digested by HindⅢ and KpnⅠ， then the lactic acid bacteria recombinant expression vector pMG36e-H5LL was constructed and cloned to obtain the recombinant plasmid containing the target genes. Results H5LL had a high possibility of being an AMPs， contained 36 amino acids， the relative molecular weight was 4 625 380，the total charge number was +10， which had hydrophilicity； there were 3 phosphorylation sites and there was no glycosylation site； H5LL belonged to intramembrane protein， without transmembrane region and signal peptide；the subcellular localization prediction results showed that the possibility of mitochondrial targeting peptide was 0.333. The α-helix junction and β-turned angle accounted for 52.78% and 22.22% in the secondary structure， and the number of α-helix was the highest in the tertiary structure. The PCR electrophoresis results of recombinant plasmid contained target gene showed a single specific band at 138 bp； the recombinant plasmid double digestion electrophoresis results showed the clear bands at 136 and 3 500 bp. Conclusion The novel heterogeneous peptide H5LL is designed and synthesized with high stability， antibacterial activity and low toxicity；the recombinant lactic acid bacteria expression vector pMG36e-H5LL is successfully constructed.

Key words: Histatin5, Antimicrobialpeptides, Bioinformatics analysis, Lactic acid bacteria, Expression vector