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Yorkshire Sow Milk Proteins:Differentially Expressed Proteins In Colostrum And Milk,Genetic Polymorphisms And Preliminary Purification

Posted on:2023-04-02Degree:MasterType:Thesis
Country:ChinaCandidate:Y J LiuFull Text:PDF
GTID:2543307022951279Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
In order to analyze the composition and variation of proteins in sow milk,a total of 50 milk samples of Yorkshire sow(n=32)collected from Xichang,Sichuan Province were analyzed for protein composition,content,and genetic polymorphisms of three kinds of proteins.A high molecular weight protein(HMWP)in milk was preliminary purified and identified.The main contents and results of this study are as follows:(1)The colostrum and milk of Yorkshire sow were studied,and the results showed that the protein content in milk was the highest on the first day of lactation(D1);Casein,immunoglobulin,β-lactoglobulin,alpha-lactalbumin,whey acid protein(WAP)and HMWP were detected in milk by SDS-PAGE,all of which changed during the lactation.(2)iTRAQ technology was used to analyze the differentially expressed proteins(DEPs)in D1,D3 and D15 sow milk.A total of 612 proteins were identified,and 243 and 384 DEPs were detected in D3 and D15 milk compared with D1,respectively.KEGG enrichment analysis showed that DEPs in D3 and D15 milk were most involved in metabolic pathway.These results revealed changes of a variety of trace protein components in sow colostrum and milk,which may reflect the changes of mammary gland functions and the requirements of piglets at different developmental stages.(3)Genomic DNA was extracted from milk,and the partial sequence of β-casein(β-CN)encoding CSN2 gene was amplified by PCR.A polymorphic locus(c.647G>A)on CSN2 gene was determined by direct sequencing,in which G allele was the dominant allele,which led to the mutation of arginine(Arg)to glutamine(Gln).According to the sequencing results of this study,the deduced β-CN sequence derived from the experimental sows did not produce casomorphin after digestion in vivo,which is different from that of bovine β-CN.(4)SDS-PAGE analysis showed that four variants of type A,B,C and D of HMWP were detected in Yorkshire sow milk,with molecular weights of 121.33±0.79 kD,109.64±0.77 kD,106.41±0.36 kD and 102.09 kD,respectively,in which B variant was predominant.There were five protein phenotypes of HMWP(AB,BB,BC,CC and CD),showing abundant polymorphisms,and AB and BC were the dominant phenotypes.The phenotype of HMWP was stable during lactation,and the expression level of HMWP in D15 milk was significantly higher than that in D1 milk.(5)SDS-PAGE combined with silver staining showed that both A and B variants of epithelial mucin(MUC1)were detected in sow milk,and the B variant was dominant.MUC1 showed three protein phenotypes of AA,AB and BB on gels,and the BB phenotypes was predominant.The phenotype of MUC1 was stable during lactation and the expression level of MUC1 in D15 milk was significantly higher than that D1 milk.(6)Crude HMWP-AB was obtained from Yorkshire sow milk by defatting,removing casein by isoelectric precipitation,and salting out with ammonium sulfate.HMWP-AB with a purity of 51.09% was obtained after High Q anion exchange column chromatography,molecular sieve chromatography and other processes.The protein may be glutathione transferase as identified by tandem mass spectrometry.
Keywords/Search Tags:Yorkshire sow, milk, iTRAQ, milk protein, genetic polymorphism, DEPs
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