Two-dimensional Electrophoresis Analysis And Immunoassay Of Bovine And Goat Milk Proteins

Bovine milk and goat milk as the most two common kinds of milk,are the important sources protein for human being.The protein differences between bovine and goat milk may not only lead to different processing characteristics,but also is the main target to distinguish bovine and goat milk.The protein of bovine and goat milk used as the main research object was to investigate the similarities and differences of protein.They were compared and analyzed by one and two-dimensional electrophoresis.The proteomic analysis method was used to identify the bovine and goat milk proteins followed by and the corresponding bioinformatics analysis.The colloidal gold immunochromatographic test strip was developed based on milk β-lactoglobulin（β-LG）.Polyacrylamide Gel Electrophoresis of bovine and goat milk were compared.Native,Reducing（SDS^R）and non-reducing(SDS^NR)conditions were used to analysis bovine and goat milk proteins,and also two-dimensional electrophoresis in Native:SDS^NR-PAGE and SDS^NR:SDS^R-PAGE condition,respectively.In the Native-PAGE pattern,the differences of bovine and goat milk proteins were mainly expressed on the β-LG band.β-LGA and β-LGB bands of bovine milk could be used as the characteristic bands to distinguish bovine and goat milk.SDS-PAGE pattern showed that the differences of electrophoretic bands between αs1-CN and αs2-CN were the main feature to distinguish bovine and goat milk with different content.One and two-dimensional electrophoresis results showed that the heat treatment would denature in heat-sensitive proteins of bovine and goat milk.The stronger heat treatment,the higher degree of protein degeneration.β-LG was easily polymerized to κ-CN and αs2-CN with disulfide bonds to form the polymers on the surface of the casein micelles and in the whey phase.Due to protein content differences of bovine and goat milk,the distribution of the polymer is slightly different.Pressure conditions also promote the binding of β-LG with casein,which were more obvious in goat milk because of the higher albumin content.Proteomics of Bovine and goat milk were analysis.The two-dimensionalelectrophoresis of IEF:SDS-PAGE were used to get the pattern of skimmed bovine and goat milk and whey proteins.The results showed that the difference of bovine and goat milk were not obvious in protein content and protein type.but the electrophoretic behavior of the same kind of protein was not similar in the2-DE map.The main different proteins between the two kind of milk in 2-DE patterns were αs1-CN,αs2-CN,κ-CN and β-LG.The main reason for these differences were that the difference in content,the presence of protein mutants and the isoelectric point difference at phosphorylation levels.The protein spots in the 2-DE gel were identified by the enzymolysis mass spectrometry,and the positions and molecular structure informations of the main casein and albumin spots in the 2-DE map were determined.Many low-abundance proteins were identified in whey proteins,such as milk serine protease,Vitamin D binding protein,α-1-antiglycoprotein and goat milk α-2-HS-glycoprotein.The results of secondary structure prediction and epitope analysis showed that the epitopes of the same kind of protein were different,and the identification of the specific epitope fragments is foundation to detect the different proteins of the bovine and goat milk protein.Research on Immunoassay Technology of bovine and goat milk were researched.The titers of antibodies α-CN-IgG,α-LA-IgG,β-CN-IgG andβ-LG-IgG prepared by using α-CN,α-LA,β-CN and β-LG as antigens were 1:204800,1: 204800,1: 3200 and 1: 25600.The competitive inhibition curves of four antibodies were determined by competitive ELISA and the result showed that the semi-inhibitory concentrations of the four antibodies were 9.2 mg/mL,1.4 mg/mL,5281.8 mg/mL and 48.4 mg/mL,respectively.As the cross-reactivity test showed that β-LG-IgG antibody had a higher specificity.The colloidal gold test strips was prepared based on β-LG-IgG.The optimal labeling pH was 8.0,and optimal protein labeling amount was 25 μg/mL.NC membrane choose Sartorius CN 140,gold pad and sample pad blocked by PEG-20000.Goat anti rabbit Ig G coating concentration was 1.0 mg/mL in C-line,while β-LG-IgG coating concentration was 1.0 mg/mL in T-line.Gold pad were immersed in 20%gold labled β-LG-IgG solution.The test strip prepared under this condition can clearly distinguish negative and positive reaction,and its detection limit of β-LG can reach 5 μg/mL.Through the electrophoretic analysis and proteomics study of bovine goat milk protein,the casein,albumin and low abundance protein in bovine and goat milk were identified.The similarities and differences of main proteins antigen epitope were analyzed and compared.Finally,the study of colloidal gold immunochromatographic test strip based on milk β-LG laid the foundation for the rapid differential detection between bovine and goat milk.