Molecular Design And Expression Of Novel Antimicrobial Peptides In Pichia Pastoris

Antibacterial peptides (AMPs), encoded by specific genes, are very small molecular peptides which are widespread in the nature. They have antimicrobial activity, and belong to the non-specific immune system of fishes, amphibians and mammals. They have wide sources, low molecular weight, and wide antimicrobial spectra. And they are not prone to drug resistance. Therefore, they have been widely applied in the fields of pharmaceutical and health care, food processing, agriculture, forestry, animal husbandry, etc.In this study, novel antimicrobials were designed and modified on the basis of natural AMPs and some reported synthetic hybrid AMPs through bioinformatics method. The method of genetic engineering was applied in order to express these novel AMPs efficiently.In this project, based on the amino acid sequences of Cecropins, Melittin, Mytilin and Buforin I, seven novel amino acid sequences of AMPs named CMM、CM1、CM2、CM3、 CM4、CM5 and bufor-J were designed by means of bioinformatics method. And their nucleotide sequences were also designed according to yeast codon preference. The corresponding target genes were amplified by PCR and cloned into different expression vectors of pPICZaA and pPIC9. Then the novel antimicrobial peptides were expressed in host strain P. pastoris GS115 and their antibacterial activities were determined.The results were as follows:① Seven novel antimicrobial peptide sequences were modified and designed by means of bioinformatics. The analysis results showed that they were in accord with antimicrobial peptides’properties.② According to the amino acid sequences and yeast codon preference, the novel AMPs target genes were amplified by PCR, cloned into pPICZaA vector, and expressed in host strain P. pastoris GS115. The results of bacteria inhibition tests showed that, for all the peptide genes cloned in pPICZaA, no bacteria inhibition was found after induced expression.③ Several AMPs target genes were cloned into pPIC9 vector, and expressed in host strain P. pastoris GS115. The experimental results showed that the expressed peptides have preliminary bacteria inhibition against Listeria monocytogenes and Staphylococcus aureus, while the antibiotic activities were not very reproducible.This study has showed that the AMPs designed by bioinformatics method had feasibility and rationality, which have laid foundation for further study about the new antibacterial peptides.