The Effect On IL-8 Gene And Protein Expression Of Xinjiang Duolang Sheep Lymphocyte By Gossypol

Xinjiang is not only a large province of animal husbandry,but also a large province of cotton cultivation.The high yield of cottonseed cake with high protein content and low price,the application of cottonseed cake as feed source is one of the effective methods to solve the shortage of feed in southern Xinjiang sheep.However,cottonseed cake contains gossypol,and gossypol is a kind of cytotoxic,vascular poison and neurotoxicity,which can cause damage to the sheep body,which restricts the application of cottonseed cake in the breeding industry.IL-8 is a chemotactic cytokine that activates neutrophils,and plays an important role in regulating inflammation and immune response.In order to understand the effect of gossypol on the immune function of IL-8 in the process of damage to Duolang sheep.The real-time fluorescence quantitative PCR method was used to determine the dynamic changes of gossypol on the lymphocytes of duolang sheep IL-8 gene at different concentrations and different times after the action,so as to guide the production practice better.The peripheral blood lymphocytes were isolated from the peripheral blood of duolang sheep.With different concentrations（5μmol/L,10μmol/L,15μmol/L,20μmol/L,25μmol/L,30μmol/L,35μ-mol/L）of gossypol.The stimulation time was 1h,2h,3h,4h and 8h,Total RNA was extracted and the real-time fluorescence quantitative PCR was used to detect the expression of IL-8 gene.The results showed that with the increase of gossypol concentration and action time,the expression level of IL-8 increased first and then decreased rapidly,gossypol concentration of 10μmol/L,2h IL-8 gene expression was the highest.In order to study the difference of IL-8 protein levels in the lymphocytes of the duolang sheep,the duolang sheep pcDNA 3.1-GFP-IL-8 eukaryotic expression vector was constructed..The IL-8 gene fragment of the lymphocyte of duolang sheep was connected with the pMD-l8T vector,and DH5a was transformed to obtain the pMD-18T-IL-8 clones.Then the pMD-18T-IL-8was identified by the method of enzyme digestion（BamHI,Xhol）and PCR detection.The results showed that the prokaryotic expression vector of pMD-18T-IL-8 was successfully constructed.After recycling the pMD-18 T-IL-8 and pcDNA3.1-GFP gene fragments of BamHI,XholI enzymes,they are linked with T₄DNA enzymes to convert DH5a,and obtained pcDNA3.1-GFP-IL-8 clones,and then identified by enzyme digestion（BamHI,Xhol）,PCR and sequence sequencing.The results showed that the pcDNA3.1-GFP-IL-8 eukaryotic expression vector was successfully constructed,the CDS region of IL-8 was cloned,the sequence length was315bp,and the IL-8 sequence of the duolang sheep and sheep was 100%by NCBI Blast comparison.We further separated the lymphocytes from the Duolang sheep,cultured 8h in vitro with RPMI1640 complete medium,then transfected pcDNA3.1-GFP and pcDNA3.1-GFP-IL-8respectively,and added them to the medium according to the Lipofectamine 2000 and plasmid 5μL,and then continued to cultivate 24h after mixing.According to the following groups:A:normal cultured 2h lymphocytes;B:10μmol/L gossypol stimulated lymphocyte cultures 2h;C:the plasmid pcDNA3.1-GFP was transfected into lymphocytes after cultured 2h;D:the plasmid pcDNA3.1-GFP was transfected into lymphocytes then used 10μmol/L gossypol stimulated cultured 2h;E:pcDNA3.1-GFP-IL-8 transfected into lymphocytes cultured 2h;F:pcDNA3.1-GFP--IL-8 was transfected into lymphocytes then used 10μmol/L gossypol stimulation culture 2h.The expression of gene and protein in IL-8 was detected by real-time fluorescence quantitative PCR and Western blotting.The relative expression of real time quantitative PCR IL-8 gene was as follows:After pcDNA3.1-GFP-IL-8 was transfected into lymphocytes then used 10μmol/L gossypol stimulation cultured 2h.of IL-8 gene and protein expression are greater than 10μmol/L gossypol stimulation lymphocytes cultured 2h of IL-8 gene expression.After pcDNA3.1-GFP-IL-8was transfected into lymphocytes then used 10μmol/L gossypol stimulation cultured 2h.of IL-8gene and protein expression are greater than after pcDNA3.1-GFP-IL-8 was transfected into lymphocytes then cultured 2h of IL-8 gene expression.The results showed that 10μmol/L gossypol stimulated lymphocyte 2h,the expression of IL-8 gene and protein are increased.The results laid the foundation for revealing the immunological function of IL-8 stimulated by gossypol.