Inant Expression And Activity Analysistone H2A Derivatives Antimicrobial Peptides

Antibiotics are drugs that fight infections caused by bacteria. Wide spread usage of antibiotics promotes the development of antibiotic-resistant bacteria. Antibiotic resistance has been regarded as one of the world’s most urgent public health problems. Identification of new antibacterial agents with high efficicay, broad-spectrum activity are required to replace traditional antibiotics. Antimicrobial peptides are an evolutionarily conserved component of the innate immune response and are found in diffferent organisms. So far, more than2000kinds of antimicrobial peptides have been reported.Hi stone-derived antimicrobial peptides (HDAPs) are identical to a part of histone subunit. HDAPs are noteworthy because it uses an uncommon mechanism whereby they kill bacteria without causing cell lysis and is able to cross bacterial membranes and enter lipid vesicles. However, it is difficult to isolate HDAPs from natural resources. In this study, we first design and synthesize the HDAPs DNA fragments ThAP and HhAP, then, GST-ThAP and GST-HhAP were expressed in E.coli and purifed by affinity chromography.The obtained results are as follows:(1) Synthesis of ThAP and HhAP DNA fragment Based on the Tetrahymena and human histone H2A sequence, HDAPs ThAP and HhAP were selected. Design and optimization of ThAP and HhAP DNA fragment genetic codon for effectively expression in E.coli. pUC57-ThAP and pUC57-HhAP were constructed.(2) Bioinformatics analysis of ThAP and HhAP The molecular weight of ThAP and HhAP, net charge, amphiphilic, isoelectric point, the secondary structure of ThAP and HhAP were analyzed by antibacterial peptide database website and SOPMA program line. The molecular weight of THAP and HhAP are2.15Kda and2.22Kda respectively; ThAP is mainly composed of a-helix structure, which accounts for57.89%and HhAP is mainly composed of a-helical (43%) and the random coil structure(38%).(3) Recombinant expression of GST-ThAP and GST-HhAP The pUC57-ThAP and pUC57-HhAP were digested by BamH I and Xhol I target gene is ligated to expression vectors pGEX-4T-1. The recombinant plasmids pGEX-4T-1-ThAP and pGEX-4T-1-HhAP were transformed into E. coli strain BL21. The recombinant engineering cell strain BL21/pGEX-4T-\-ThAP and BL21/pGEX-4T-1-HhAP were obtained. GST-ThAP and GST-HhAP were expressed at37℃with0.1mM IPTG induction for four hours in BL21/pGEX-4T-1-ThAP and BL21/pGEX-4T-1-HhAP, respectively. GST-ThAP and GST-HhAP were purified using GST SePharose high Performance affinity column.(4) Antibacterial activity analysis of recombinant antimicrobial peptides GST-ThAP and GST-HhAP. The growth rate of gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus was inhibited with the treatment of GST-ThAP (ThAP) and GST-HhAP. GST-ThAP lead to growth inhibition of E. coli which was much more effective to GST-HhAP. Furthermore, the scratch assay was used to observe the impact of GST-ThAP, GST-HhAP and ThAP on the glial cell line C6migration. The results show that migration speed are similar at8h cultivation, and the migration rate is Tris> GST-the ThAP (5uL)> GST-ThAP (15uL)> ThAP (5uL) after20h cultivation.In summary, antimicrobial peptides ThAP and HhAP, derived of Tetrahymena and human histone H2A, were constructed and expressed in E. coli successfully. GST-ThAP and GST-HhAP inhibit growing of gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. The results indicated the ThAP and HhAP are valuable for antibacterial drug exploration and exploitation.