Isolation, Purification And Indentification Of Lactoferrin And Expression Of Recombinant Chinese Miniature Pig Lactoferrin Gene In Pichia Pastoris

The lactoferrin as an iron-binding glycoprotein, is presented in numerous exocrine secretions of mammals, including milk, tears, saliva, et al.Milk,especially porcine colostrums contains lactoferrin at a much higher level than others. Lactoferrin is a protein with multi-functions,including broad spectrum antibacterial properties, antiviral activity, anticancer activity and immunoregulatory activity.The methylotrophic yeast Pichia pastoris has been developed as an efficient expression system for foreign proteins. It can grow on methanol as a sole carbon and energy source. As a eukaryotic expression system, Pichia pastoris has many advantages over prokaryotes and other higher eukaryotes. It is as easy to manipulate and culture as prokaryotes and is able to process posttranslational modifications as other higher eukaryotes.In this experiment,LF was separated and purified by 3 times salt-out and 1 time gel chromatography from porcine colostrums, and measured by SDS-PAGE. For the acquired lactoferrin, the molecular weight was estimated at 80kDa , and the purity was about 91%. Antibody against LF was achieved by injection of rabbits and chicken with LF.Lactoferrin gene of Chinese miniature pig was amplified using RT-PCR, the PCR products were inserted into pMD19-T vector routinely, and the positive recombinants were identified by endonuclease digestion,PCR and DNA sequencing. Sequence analysis found 99% homology with other sequences in GenBank. Digested with XhoI and XbaI, a fragment 2102bp of the PCR product was cloned into Pichia pastoris expression vector pPICZαA containing the inducible promoter of the alcohol oxidase-1 gene(AOX1) and theα-mating factor signal peptide. The recombinant plasmid of pPICZαA-pLF was lineamized by Sad and transformed into GS115 by electroporation. The mufti-copy insertion transformants were screened by Zeocin-resistance and induced by 0.5% methanol. The Pichia pastoris culture medium was concentrated by TCA/DOC and characterized using SDS-PAGE and Western blot. The results showed that the rPLF with a molecular weight of approximately 80kDa which was similar with nature pLF was well expressed in Pichia pastoris.