Heterologous Expression Of Cecropin AD In Pichia Pastoris

Antimicrobial peptides(AMPS)are an important part of the innate immune system of most organisms.They are a kind of small peptides with antibacterial activity,which can effectively kill pathogenic bacteria and resist the invasion of foreign bacteria and viruses.Cecropin AD is a kind of cecropin antibacterial peptide.Cecropin is the most effective antibacterial peptide at present,and its industrial production lays a foundation for its application in agriculture and aquaculture.In this study,we used AOX and GAP to construct inducible gene engineering strain and constitutive gene engineering strain,and finally selected constitutive expression strain as the follow-up experimental strain to study the high-efficiency expression of cecropin AD in Pichia pastoris and the optimization of fermentation conditions,and tested its characteristics.1.In this paper,we synthesized the cecropin AD gene fragment and amplified it with Xho I and Not I restriction site by PCR.Connect it with p PIC9K,which digested by same enzymes,transfer it into the JM109 competent cell,select the transformants to extract the plasmids,then transfer the reconstruct plasmids p PIC9K-CAD into Pichia pastoris GS115,select the positive clones.Finally,two genetically engineered strains GS115/p PIC9K-CAD-5 and GS115/p PIC9K-CAD-9 with better antibacterial effect were obtaine.These strains were preserved by glycerol.2.Using primers to amplify cecropin AD gene with EcoR I and Not I restriction site.Connect it with p GAPZ?A,which digested by same enzymes,transfer it into the E.coli competent cell,then the linearization plasmid p GAPZ?A-CAD was transformed into Pichia pastoris X33.The gene engineering strain X33/GCAD-3 was obtained.The constructed plasmid PUCGAP-CAD was transformed into the electric shock receptive state of X33/GCAD-3,and finally multicopy strain X33/GUCAD-3 were obtained.The strain was used as the subsequent experimental strain.3.Tricine-SDS-PAGE and LC-MS were used to identify the fermentation products of X33/GUCAD-3.The results showed the product with antibacterial activity was cecropin AD.The YDFM,YPD and BMGY were used as the fermentation media.The results showed that the most suitable fermentation media was YDFM.Glycerol,glucose and maltose were used as the sole carbon source,and the most suitable carbon source was glycerol.The condition of the nitrogen source addition and the initial pH in medium was optimized and the results showed at 24 h and 48 h,5 g/L organic nitrogen source was supplemented respectively,and at the initial pH of 7.0,the bacteriostatic effect was the best,and its biological activity was 8.84×10~8 U/L.4.The antibacterial spectrum of cecropin AD was determined,which showed that it had broad antibacterial spectrum,and had antibacterial activity against Gram-positive and Gram-negative bacteria,but no effect on fungi.Cecropin AD has antibacterial activity against some common pathogenic bacteria.High temperature resistance is strong,and it can still have high antibacterial activity when it is placed at 100?for3 h.When treated with 30 U/ml pepsin and protease K respectively for 2.5 h,the antibacterial activity of cecropin AD slightly decrease compared with cecropin AD without protease treatment.Cecropin AD still had a certain antibacterial activity unde30 U/ml trypsin protease,but compared with cecropin AD without trypsin treatment,its antibacterial activity obviously decrease.More over,the hemolytic activity of cecropin AD was not found.