Expression Of Antimicrobial Peptides From Fish In Pichia Pastoris And Their Bioactivities

In recent years, the overuse of antibiotics has led to some consequences such as dysbacteriosis in vivo and the generation of resistant strains, causing public health and food safety issues, so we urgently need to find a new antibacterial drugs to alleviate these problems. When organism is invaded by pathogens, the antimicrobial peptides which have immune antimicrobial activity will be synthesized rapidly. Also Antimicrobial peptides have many advantages, such as the stability of structure and activity, no drug resistance, no drug residues, no environmental pollution, etc., they have received more and more attention. Fish lives in water environment containing a variety of microorganisms, frequently in contact with a large number of pathogens. Fish has a well-developed non-specific immune system. Antimicrobial peptides are an important part of the non-specific immune system of fish. When fish is damaged or invaded by pathogens, the antimicrobial peptides are able to be generate rapidly and spread in the fish body. This paper mainly studies two kinds of fish antimicrobial peptide, one is hepcidin of the channel catfish(Ictalurus punctatus) and tilapia(Oreochromis niloticus), the other is hipposin which comes from the N-terminal derivative of histone H2 A of Atlantic halibut(Hippoglossus hippoglossus L.).Hepcidin is small cationic peptide with antibacterial activity and regulation of iron metabolism expressed mainly in the liver of living organisms, and it plays important role in the host’s immune response against microbial invasion and the regulation of iron metabolism. Hepcidin has a high structural conservation among species, which is composed of signal peptide, prodomain and mature peptide, especially the mature peptide region contains 8 cysteine residues which form 4 disulfide bonds in the space. The structure has a close relation with structural stability and biological activity. Hepcidin was originally isolated from human serum, after that we found hepcidin in many fishes, but there is few report about recombinant expression of hepcidin of fish.Hipposin is N-terminal derivative of Atlantic halibut histone H2 A. Hipposin was isolated from the skin mucus of Atlantic halibut by Birkemo. The structure of hipposin is consist of α-helix and random coil. It contains 51 amino acids that include 15 basic amino acids and no acidic amino acid. To date, few studies focus on hipposin, much less recombinant expression.The study mainly carried out the expression, the purification and activity detection research of hepcidin and hipposin from fish, including the following five parts:The first part of this study focuses on the connection of c DNA of hepcidin’s mature peptide from two fish and the construction of recombinant expression vector. The existing hepcidin mature peptide c DNAs from channel catfish(m CH) and tilapia(m TH) were used as templates, which were connected by SOE-PCR. Their primers contain cross complementary sequences. Then the series fragment m CH-m TH was used as template, and the primers were designed with Eco R I and Not I restriction sites to amplify the sticky ends. m CH-m TH and expression vector p PIC9 K were digested by Eco R I and Not I restriction sites. Connected the digested m CH-m TH and expression vector p PIC9 K by T4 DNA ligase. Colony PCR test, double enzyme digestion and sequencing confirmed that p PIC9K-m CH-m TH recombinant plasmid was successfully constructed.The second part of this study focuses on the expression and purification of m CH-m TH in Pichia pastoris strain GS115. The p PIC9K-m CH-m TH plasmid was linearized by Sac I, then it was transformed into P.pastoris GS115. His+Mut+ high-copy clones were screened by the MD plate without histidine, the YPD plate with different concentrations of G418, MD and MM plate. The yeast genome was extracted to verify whether the target gene was integrated into the yeast chromosome. The screened strains were cultivated in BMGY medium, then transferred to BMMY medium at 30℃ for shaking culture. The target protein was induced by 1% methanol. We found that the most appropriate expression time was 72 h. The 72 h fermentation supernatant was used to separate and purify the target protein by SP-Sepharose cation exchange chromatography. The expression yield of m CH-m TH is 77mg/L by using folin-phend method.The third part of this study focuses on the identification of antibacterial activity of m CH-m TH. By agarose diffusion method, we found that the 10-fold concentrated fermentation supernatant has good inhibitory effect on Gram-positive S. aureus and gram-negative P. aeruginosa, we could see the clear inhibition zone. By broth dilution method, we found that the purified protein m CH-m TH has strong antibacterial effect on S. aureus, P. aeruginosa, E. coli, B. cereus, L. monocytogenes, S. enterica.The fourth part of this study focuses on the synthesis of hipposin gene and the construction of recombinant vector. According to the codon preference of P.pastoris, we optimized the codon of hipposin gene. Then we added the coding sequence of signal peptidase Kex 2 recognition site at the 5’ end, six histidine codons at the 3’ end and Xho I and Xba I restriction sites at both ends. After that we synthesised the optimized gene. The synthesized hipposin gene(HIP) and p PICZαA were constructed combinant expression vector p PICZαA-HIP, we detected whether the recombinant expression vector was constructed successfully by double digestion. The gene sequencing was used for further verification. The constructed recombinant expression vector was linearized by Sac I, then it was transformed into P.pastoris strain X-33. High-copy clones were selected by Zeocin resistant. Their yeast genomes were extracted to verifiy that HIP has been integrated into the yeast chromosome successfully.The fifth part of this study focuses on the expression and purification of hipposin in P.pastoris. Screened strains were cultured in BMGY, then transferred to BMMY medium at 30℃ shaking culture, induced by 1% methanol. The expression of hipposin reached the highest expression level at 96 h. Western Blot analysis further showed that the target protein hipposin was expressed. The fermentation supernatant was collected at 96 h, which was used to separate the target protein by immobilized metal affinity chromatography(IMAC) in the protein purification systems.