Expression, Purification And Antimicrobial Activity Analysis Of A Recombination Catfish Antimicrobial Peptide Parasin Ⅰ In Escherichia Coli

The use of low levels of antibiotics as growth promoters in animal feeds and the extensive use of antibiotics to treat animal infections are thought to be the cause of an alarming increase in antibiotic resistance among Gram-negative, Gram-positive, fungal pathogens and antibiotic residual in livestock products, a highly debated and controversial issue. Bacterial resistance to many classes of antibiotics is becoming a major clinical problem. Therefore, it is necessary to find an antibiotics that are active in vivo, are fast acting and broad-spectrum, do not induce bacterial resistance and have limited side effects. Antimicrobial peptides (AMPs) are good candidates. A large variety of AMPs are found in animal. AMPs, also known as host defense peptides, play major roles in the innate immune system, and protect against a wide variety of bacterial, fungal, viral, and other pathogenic infections. Recently, a potent AMPs named parasin I was isolated from the skin mucus of wounded catfish. Parasin I showed good activity against different Microorganisms, including Gram-negative and Gram-positive bacteria and fungi and no hemolysis. So Parasin I possess potent value of research and development. But natural parasin I was expressed by epidermal injure induced. It is difficulty to isolate and purify ParasinⅠthe from the skin mucus of wounded catfish. The above of all limited its exploitation.The objective of this study was to develop an efficient expression system in Escheri-chia coli to produce a recombinant AMPs Parasin I with high yield by genetic engineering.A length of the 57bp gene sequencing of catfish Parasin I was designed, optimized and synthesized according to its peptide sequence and Escherichia coli codon favor and ligated to a cloning vector pMD18-T. At the same time, the upstream primer of Parasin I gene which designed by taking the synthesized gene as the template, included endonuclease site of Activated Factor Xa. After sequencing, the sequence both synthesized and cloned are consensus.The pMD18-T-paraⅠwas digested by KpnⅠand HindⅢ, purpose gene is recycled and ligated to vector pET32-a(+) which is an efficient expression system in Escherichia coli and digested by same method to construct recombinant plasmid pET32-a(+)-Para I. The pET-32-a(+)-Para I was delivered into Rosetta(DE3) strain by lightning stroke. Positive trans-formants was screened by Ampicillin and expressed at 37℃and 20℃by 1PTG induced. The quantity of fusion protein was estimated using protein gelatin analysis software (an analysis that one 4.6.2). The proportion of two kinds of protein is 49.7%and 48.9% of total protein, respectively. Inclusion body was denaturalized in 8M urea and separated through dislysis. The solubility protein containing a 6×His Tag appended to the C-terminus was purified using Ni Sepharose high Performance affinity column. Coomassie Brilliant Blue determined that the concentration of protein is 0.179mg/mL.The purified fusion protein was restored to natural AMPs Parasin 1 by digesting with factor Xa to remove the Trx-tag and his-tag fusion tag. Agar hole diffusion method deter-mined antimicrobial activity of Parasin I to Staphylococcus aureus of the logarithmic phase taking Amp as the control. The results showed that Amp (5μg) can show a strong antimic-robial activity, restored Parasin I could inhibit the growth of Slaphylococcus aureus in the concentration of 36μg, unrestored fusion protein had no activity.In short, the successful expression of catfish AMPs ParasinⅠgene in Escherichia coli in this study would provide an efficient and facile platform for the production or study of its exploitation.