Effects Of BLV On Milk Composition Synthesis Function In MAC-T

Bovine leukemia virus(BLV)is a member of retrovirus family,it can establish persistent infection in bovine and most infected cows have no obvious clinical symptoms or long-term latent infection.BLV is widely prevalent in the world.It can infect cow mammary gland through blood and lymphatic system.At present,it is known that BLV can replicate stably in mammary epithelial cells of dairy cows.Lifetime lactation,lactoferrin content,milk fat and total milk protein yield of BLV positive bovine are reduced.However,the mechanism of BLV infection on milk protein and milk fat synthesis is not clear.In this study,we first rescued a single source of BLV,constructed a model of BLV infected bovine mammary epithelial cell line(MAC-T),and studied the effect of BLV infection on milk composition synthesis in MAC-T,so as to provide a research basis for a comprehensive understanding of the effect of BLV down-infection mammary glands on milk quality.106 lactating cows in a large-scale pasture in Daqing city were selected as the research objects to analyze the effects of BLV positive and negative cows on milk yield,milk fat and milk protein content.The infectious clone containing full-length BLV gene was used to infect 293-T cells to rescue BLV;the infectivity of rescue virus in 293-T cells and MAC-T cells was calculated by Reed-Muench method;the co culture system of BLV + 293-T + F81 was established to verify the rescue virus strain.The co-culture system of BLV + MAC-T was established,and the growth curve of BLV in MAC-T was drawn by RT-PCR.MAC-T was infected with BLV at a dose of 3 MOI.The levels of fatty acid synthase(FASN),acetyl-Co A carboxylase1(ACC1)sterol regulatory element binding protein(SREBP)and(peroxisome proliferators-activated receptors(PPARγ)in MAC-T at different time points(6 h,12 h,24 h,36 h,48 h and 60 h)were detected by fluorescence quantitative PCR,and the relative mRNA expression levels of four caseins,namely,beta casein(CSN2),casein alpha S1(CSN1S1),kappa casein(CSN3)and casein alpha S2(CSN1S2),were measured;Western blot was used to detect the protein expression levels of SREBP1,PPARγ,ACC1,FASN and CSN2.The formation of lipid droplets was detected by laser confocal microscopy.After BLV infected MAC-T with 1 MOI,the cells were subcultured,then the first,fourth,sixth and eleventh passage cells were detected to determine the effect of long-term chronic BLV infection on the milk composition synthesis of breast epithelial cells.The results showed that: BLV infection had no significant effect on milk yield,milk fat content and milk protein content decreased significantly,but the difference was not significant.The BLV strain was successfully rescued in 293-T cells,the infectivity of it in MAC-T was 58.75TCID50/m L.the logarithmic growth period ended 48 hours after BLV infected MAC-T.BLV persistent infection highly significantly reduced the transcription,translation and nuclear shift levels of SREBP1(P < 0.001),ACC1(P < 0.001)and FASN(P < 0.001),and highly significantly increased the translation level of PPARγ(P < 0.001);After BLV infection,the mRNA expression levels of CSN2,CSN3,CSN1S1 and CSN1S2 in MAC-T were highly significantly increased(P <0.001),but the expression level of casein was highly significantly decreased(P < 0.001).MAC-T cells were subcultured after BLV infection,SREBP1 mRNA expression level was significantly decreased(P < 0.05),and PPARγ mRNA expression level had no biological difference;Consistent with the results of mRNA expression,the protein expression level of SREBP1 was highly significantly decreased(P < 0.001),and there was no biological difference in the protein expression level of PPARγ;The mRNA expression levels of FASN and ACC1 decreased highly significantly(P < 0.001),however,with the increase of cell passage,the mRNA expression levels of FASN and ACC1 gradually recovered.Different from the mRNA expression level,the protein expression level of FASN gradually decreased to a very significant difference(P < 0.001),and the protein expression level of ACC1 increased highly significantly(P < 0.001);MAC-T cells were subcultured after BLV infection,the relative mRNA expression levels of milk protein related genes CSN2,CSN3,CSN1S1 and CSN1S2 were highly significantly increased(P < 0.001),and the mRNA expression level gradually increased with the increase of passage number,but the protein expression level of CSN2 gradually increased with the increase of passage number.In conclusion,BLV infection can inhibit the expression of CSN2 in MAC-T,inhibit the transcription and translation of lipid metabolism related molecules in MAC-T,and affect de novo fatty acids(FAs)synthesis,thus regulating milk fat synthesis.