Prokaryotic Expression And Initial Antibiotic Activity Identification Of Hybrid Peptide HMCM

Antimicrobial peptides are a class of micromolecule peptides which show positive charge and amphipathicity. They have many advantages, such as broad antimicrobial spectrum, not easy to produce resistance, and so on. The sources of antimicrobial peptides are very wide and the classifications of antimicrobial peptides are various. Antimicrobial peptides have very strong biological activity, so they are widely used in pharmaceutical fields, animal husbandry, food and other fields.Hybrid peptide is one of the products of genetic engineering techniques. Using the genetic engineering technology to produce antimicrobial peptides has the advantages of short time and high content. Hybrid peptides are mainly composed of some parts of two or more antimicrobial peptides, with the advantages of broad- pectrum, low toxicity and high efficiency. Studies show that certain modified peptides have similar or even stronger biological activity and lower toxicity with the comparison of prototype. There is great research value of hybrid peptide.In this topic, the effective segments of mammalian antimicrobial peptides (Hexap- eptide), amphibian antimicrobial peptides (Magainin), and insect antimicrobial peptides (Cecropin A and Melittin) were chose to build a new and more efficient hybrid peptide HMCM. In this study, the gene sequence of target protein HMCM was designed based on the amino acid sequence of target peptide HMCM and the codon bias of E. coli B cells, and the bioinformatics of HMCM was analyzed. To acquire the target gene, three primers were designed to execute two rounds of PCR reaction. Then, the target gene was transferred to prokaryotic expression vector pET-32a-c(+) and the recombinant plasmid named pET-32a-HMCM was constructed. And then, the recombinant plasmid was converted into the prokaryotic expression cells E.coli BL21 (DE3) and the fusion expression was achieved. After fumbling the inductive time, IPTG concentration, OD600 and temperature, the best expression conditions of fusion protein were ensured. It was found that the fusion protein existed as soluble form. After that, the fusion protein was purified quickly by affinity chromatography and identified by western-blotting. At last, the fusion protein was digested by EK enzyme and the protein HMCM was released, and the initial bacterinertness of HMCM was analyzed.The main results of this study were as follows: ①Bioinformatics analysis showed that HMCM had the basic properties of antimi- crobial peptides: The molecular weight was 4.07 kD, and the isoelectric point was 12.805. The N terminal was hydrophilic, and the C terminal was hydrophobic. The secondary structure was mainlyα-helix structure, with a small quantity ofβ-sheet structure,β-turn, and random coil.②The target gene which encoded HMCM was acquired after two rounds of PCR reactions. After that, the gene was transfered to the prokaryotic expression vector pET- 32a-c(+) digested by Kpn I and EcoR I, and the recombinant vector pET-32a-HMCM was obtained .③The test expression of fusion protein in E.coli BL21 (DE3) and the optimization of expression conditions were achieved. The ultimate optimal expression conditions were as follows: The inductive time was 3 h, the initial inductive OD600 was 0.9 or so, the inductive IPTG concentration was 0.8 mmol/L, and the inductive temperature was 30℃.④The ultrasonic fragmentation experiments showed that the fusion protein existed in the cell supernatant as soluble form. After ultrafiltration and affinity chromatogram- phy, a single target band was obtained and the fusion protein was identified by western- blotting.⑤The measured result of protein concentration showed that the expression level of fusion protein reached milligrams level. 11.92 mg fusion protein was acquired per liter of bacterial liquid.⑥The fusion protein was digested by EK enzyme and HMCM was released. The result was very well.⑦The initial antibacterial activity of HMCM was identified by bacillus subtilis and E.coli DH5αbacteria. The results showed that HMCM obtained by digestion and the fusion protein not digested could inhibit the growth of bacillus subtilis, and HMCM was stronger than fusion protein. However, HMCM and the fusion protein couldn't inhibit the growth of E. coli DH5α.