Construction Of Recombinant Expression Vector Of Sea Bream And Its Expression In Yeast Pichia Pastoris

Antimicrobial peptides have been identified in various species ranging from bacteria, frogs to mammals, including humans. They form the first line of host defence against pathogenic infections and are a key component of the ancient innate immune system. Our research mainly focuses on the construction of recombinant sea bream hepcidin and pleuocidin expression vector and the expression of recombinant hepcidin in Pichia pastoris.Up to now, a 100bp hepcidin fragment and a 1 )0bp pleurocidin fragment have been amplified separately with PCR method. pPBSKII -hepcidin and pPBSKII -pleurocidin cDNA are used as templates. Ligation using T4 ligase is performed between the cohesive ends of the xho I and the xba I using a 1:3 molar ratio between vector and insert, and is conducted at 16 C o/n. Ligation mixture are transformed into competent E. coli and selected on plates. Transformants are isolated and analyzed for the presence and orientation of insert. Restriction analyses, using the endonuclease xho I , xba I , BamH I , and PCR methods are performed to confirm recombinant plasmid pPICZ a A- rsbHEPC and pPICZ a A- rsbPLEU.The plasmids pPICZ a A- rsbHEPC and pPICZ a A- rsbPLEU, previously amplified in E.coli DH5 a and purified by the QIAGEN Plasmid Midi Kit(100), are linearized by sac I and used to transform eletrocompetent yeast X-33 strain. Spread 10, 25, 100 and 200 1 each on separate labeled YPDS plates containing 100ng/ml zeocin. Plating at low cell densities favors efficient zeocin selection. Then the plates were left at 30C from 3 to 7 days until colonies form without shaking. Because X-33 is used as the host, the transformants should be Mut+. However, with the presence of the AOX1 sequences in the plasmid, there is a chance that recombination will occur in the 3'AOX1 regior also, disrupting the wild-type AOX1 gene and creating Muts transformants. 10-20 zeocin-resistant colonies are picked to confirm the Mut phenotype. PCR is also used to analyze for t le presence of insert. Several Mut+ and Muts recombinant strains are incubated under the induction of methanol. Analyze the supernatants and cell pellets for protein expression by silver-stained SDS-PAGE and functional assay. The antibacterial effects of antimicrc bial peptides on the E.coli DH5 a andV. anguillarum are performed. The result shows that recombinant hepcidin has antibacterial effects on E.coli DH5 a ,while it has no effects on V. anguillarum.