Studies On The Separation And Purification And Biological Activity Of Transgenic Goat Of RhLF In The Milk

Human lactoferrin (hLF), an iron-binding glycoprotein, has many kinds of biological activities. hLF based on the unique physical and chemical properties and biological functions, has been widely used in food, medicine and feed additives. The amount of hLF required for extensive or widespread demand is limited due to the lack of supply and pathogenic contamination during processing. The mammary gland bioreactor Compared to these protein expression systems has potential to produce biologically active proteins in large quantities at a relatively low cost. Because of this, The use of genetic engineering techniques to develop recombinant human lactoferrin (rhLF) has become a hot research and development trends.In previous studies, our laboratory had constructed rhLF mammary gland-specific expression vector containing hLF cDNA and got transgenic dairy goats. At the same time, we chose two rhLF transgenic dairy goats’milk (L6-4, L3-1) for samples. After purifying the rhLF from the milk, we studied that whether rhLF had the same bioactivities with hLF or not.After collecting the milk of transgenic dairy goats L6-4,L3-1, the whey was acquired by wiping off fat and casein. The whey which was thrice through the ammonium sulfate precipitation obtained the final precipitate. the precipitate was dissolved in ultrapure water and then concentrated to30mg/ml with PEG20000after dialyzed use the PBS (PH7.4) for dialyzate by24h. Using the Western-blotting assay to analyze. The concentrated solution was diluted to20mg/ml and then extracted the rhLF by gel filtration chromatography with sampling volume7ml, the flow rate of0.5ml/min,buffer PH value of7.4.After Western-blotting assay, the filter paper (6.0mm diameter)with the extractive at the following concentration (2mg/ml) of10μL was added at37℃for14-16h to observe inhibition zones. The rhLF was mixed with Tris-HCL buffer, NaHCO3and Fe2(SO4)26H2O. The subsequent changes in absorption were monitored within10min at25℃in order to assess the iron-binding ability of rhLf by UV spectrophotometer.The results of Western-blotting showed that rhLF could specially bind with rabbit anti hLF antibody and had the same molecular weight as the hLF. The rhLF has been isolated in the first elution peak. The rhLF and hLF had the same antibacterial activity with the same concentration of protein. Studies of iron-binding showed that rhLF and hLF had the same iron-binding activity because of the same absorption peak at465nm. The research indicated that the rhLF which was extracted from the milk of transgenic goat had the same molecular and biological activity as the hLF. All of them were the basis for large-scale rhLF production.