Expression In Pichia Pastoris, Physicochemical And Biological Identification Of Bovine IFN-α0, IFN-α2,IFN-α0/LFB

Interferon(IFN) is a kind of multfunctional glycoprotein with high antiviral, antitumor and immunomodulatory activity. IFN-α is the most active antiviral cytokine currently known, it’s not direct antiviral substances but inhibit viral reproduction by antiviral protein(AVP) which is activated by interferon receptor(IFNAR) on the cell membrane of target cells. Bovine α-interferon(Bo IFN-α) is one of the IFN-α, the systematic research of Bo IFN-α is important.Lactoferrincin is an active peptide separated from the N-terminal region of bovine lactoferrin, it’s amino acid sequence corresponding to bovine lactoferrin first 17-41 amino acids. Experiments show that lactoferrincin can kill or inhibit the growth of a variety of gram-positive bacteria and negative bacteria including E. coli and Staphylococcus aureus. The antibacterial effect is lethal, most tests confirm that Lactoferrincin can lead to rapid loss of colony forming ability of bacteria. In addition to antibacterial activity, Lactoferrincin also has antiparasitic, antitumor, antifungal, antiviral and immunomodulatory effect.In this study, we use the PCR method to obtain the mature peptide gene of Bo IFN-α0/2 from recombinant plasmid containing the open reading frame of Bo IFN-α0 and Bo IFN-α2 which were prepared and save by our laboratory. Recombinant proteins Bo IFN-α0/2 were secreted expression in the yeast GS115, the physicochemical properties and biological activities of interferon proteins were studied. Applying two different vectors(p PICZαA and p GAPZαA), derivatives of LFB(LFcin B15-W4,10) and Bo IFN-α0 were tandem expressed in yeast GS115. The the antiviral activity of three fusion protein were detected on VSV/MDBK system and the antibacterial activity against Staphylococcus aureus ATCC25923 were detected in MH broth.Studies show, Bo IFN-α0 and Bo IFN-α2 ant-VSV activity in MDBK cells were: 5.71×106U/mg and 1.36×106U/mg, anti-IBRV activity were: 7.31×104U/mg and 3.78×104U/mg. Physical and chemical properties analysis showed Bo IFN-α0/2 are very sensitive to trypsin, not sensitive to temperature, but acid resistance. It is typical of IFN-α. Three fusion protein: Bo IFN-α0-LFB(p PICZαA), Bo IFN-α0-LFB(p GAPZαA) and LFB-Bo IFN-α0(p GAPZαA) antiviral activity on VSV/MDBK system were: 2.04×106U/mg, 1.83×106U/mg and 1.56×106U/mg, in MH broth they can not inhibit the growth of Staphylococcus aureus ATCC25923.The results of our research together with the results of our laboratory studies of the other researchers composed of bovine interferon-α family, while we proof lactoferrincin targets within bacterial cells instead membrane of bacteria from the side.