Studies On The Expression Of Human Beta-defensin-2 By Heterologous Recombinant

Compared with traditional antibiotics, antimicrobial peptides have many advantages such as broad spectrum antimicrobial, not easy the creation bears the medicine, good thermal stability etc. Therefore it is expected to become a new generation of anti-infection drugs. Human ?-Defension-2 (HBD-2) is a kind of small molecule alkaline cationic antimicrobial peptide that mainly exist in the infection of mouth mucosa and skin tissue the oral mucosa. Meanwhile, it is trace expressed in healthy keratin, oral cavity, and intestinal system. HBD-2 can kill the bacteria, fungus and other pathogenic microorganisms as the body's defense. It also can inhibit the growth of tumor cells. So it has vast of potential applied outlook in medicine,food and numerous fields.This study consults the codon usage of the original HBD-2 in E.coli at first. The HBD-2 gene was optimized and inserted into the expression vector pET32a by reference to the codon preference of E.coli. The recombinant strain of HBD-2 was constructed by transforming E.coli Rossita host strain. Results show that the mature HBD-2 recombinant protein could successfully express in soluble form. The protein concentration was 0.504 mg/ml after purification by his-tag affinity chromatography. The results of antibacterial test showed that the recombinant protein had antibacterial activity against Escherichia coli and Staphylococcus aureus,but had no antibacterial activity against lactic acid bacteria WCFS1 strain. The results are consistent with previous reports.Considering that recombinant HBD-2 protein has antibacterial activity against E.coli,we presumed that selection of E.coli to make host strains have limitations. Therefore, this study continues carrying out heterologous expression of recombinant HBD-2 protein in lactic acid bacteria. The inducible and constitutive expression systems were selected, but the target protein was not detected in the supernatant of the recombinant cells. Furthermore,a gene expression strategy was developed. Due to construction of 2-8 copies of the mature HBD-2 gene in series expression of the strain, we used the Western-blot detect the target band in the recombinant supernatant of the cells, which showed that the expression of mature HBD-2 protein was successfully expressed in the supernatant. But the subsequent antibacterial activity did not detect the antibacterial activity of the protein.Although the gene operation is difficult in lactic acid bacteria expression system and the protein expression is low, the safety of lactic acid bacteria host resulting in broad prospects. The target protein in this subject has antibacterial activity against E. coli host,but not for lactic acid bacteria. Therefore, we choose lactic acid bacteria expression system to study the heterologous expression of the protein, with a positive significance in the application of lactic acid bacteria system.