Expression Of TH1-5 Mature Peptide In Pichia Pastoris And Analysis Of Biological Functions

Hepcidin is an important class of antimicrobial peptides with broad antimicrobial spectrum and unique mechanism of action. In recent years, tilapia is important species for export in china, but it was suffered from serious bacterial infection. Under the condition of increasing trend of aquatic pathogens drug resistant, the research of recombinant expression of tilapia hepcidin, can provide a reference for the development of new antibacterial drugs. In our study, the tilapia hepcidin (TH1-5) was expressed in secretion manner by Pichia pastor is GS115. Our results are as follows.(1) Expression of TH1-5 in pichia pastorisThe synthetic cDNA of TH1-5 mature peptide with the restriction enzyme cutting sites of EcoR Ⅰ and Not Ⅰ, which was optimized according to the codon of Pichia pastoris GS115, was cloned into the PUC57-simple vector. The cloning vector of the PUC57-DNA-simple-THl-5 was digested by EcoR Ⅰ and Not Ⅰ. The fragment with the cDNA of TH1-5 was cloned into the pPIC9K vector. After identification with PCR and sequencing, the pPIC9K-THl-5 was lineated by SacⅠ or Bgl Ⅱ, and was electricity transformed into the Pichia pastoris GS115. The positive clone (YH3) was induced with methanol, and the supernatant of induced product was freeze-dried.(2) Establishment of dot-ELISA method for detection of TH 1-5Dot-ELISA for detection of TH 1-5 was established with synthetic TH1-5 as a standard (positive control), rabbit anti-THl-5 antibody as first antibody, HRP-labelled goat anti-rabbit IgG. The optimum conditions of dot-ELISA as follow; the optimal dilution of first antibody and secondary antibody was 1:50 and 1:200, respectively. We detect TH1-5 in expression supernatant of the positive clone (YH3) through the dot-ELISA method.(3) Effect of recombinant expressed TH1-5 on pathogen and peripheral blood lymphocyteThe expression supernatant of positive clone (YH3) with recombinant expressed TH1-5, demonstrated obvious antibacterial activity on Escherichia coli (CMCC44102), Stapylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853) and Enterococcus faecal is (ATCC29212).The expression supernatant of positive clone (YH3) with recombinant expressed TH1-5 at the concentration range from 0.2 to 6.25 mg/mL, can promote the proliferation of chicken peripheral blood lymphocyte, but the difference is not significant compared with the expression supernatant of empty vector (PO) at the same concentration. In the synergy of ConA at 0.125 μg/mL. the proliferation of lymphocytes under the stimulation of the supernatant of YH3 with recombinant THI-5 (0.2～1.6 mg/mL). is stronger than that under the stimulation of supernatant of PO at same concentration.In summary, the expression vector pPIC9K-TH1-5 is successfully constructed. The recombinant Pichia pastoris can express TH1-5. The recombinant expressed TH1-5 can be detected by Dot-ELISA. Recombinant expressed TH1-5 demonstrated obvious antibacterial activity and can promote the proliferation of chicken peripheral blood lymphocyte in the synergy of ConA.