| Enterotoxigenic Escherichia coli F18(E.coli F18)is the main pathogenic that causing post-weaning piglets diarrhea,which is an important threaten factor in pig industry.The pathway of E.coli F18 receptor synthesis and inflammatoty immmue signaling regulate the resistance of weaned pigs to E.coli F18.Lipopolysaccharide-binding protein(LBP)not only bind to lipopolysaccharide(LPS)major component of Gram-negative bacteria cell wall,activate the immune signaling pathways,but also neutralize the LPS and then reduce biological activity of LPS.LBP plays a key role in immune defense mechanisms in vivo.LPS is an important pathogenic factor of E.coli F18,as Gram-negative bacteria.Therefore,LBP might take part in the resistance of weaned pigs to E.coli F18.This experiment use Meishan pigs as test object to explore the LBP expression patterns and methylation regulating mechanism,which will provide a certain experimental and theoretical basis for knowing the LBP function and further applying this gene in pig disease-resistant breeding.This experiment detected the LBP mRNA expression of various tissue in 35 days-old Meishan piglets and achieve tissue expression profile include heart,liver,spleen,lung,kidney,stomach,muscle,thymus,mesentery lymph node,duodenum,jejunum and ileum.We detected LBP expression of the liver at various development stages(1,7,14,21,28,35 and 158 days-old).Furthermore,we detected the LBP expression in pig small intestinal epithelial cell(IPEC-J2)before and after various concentrations LPS stimulation for 2h,4h and 6h,respectively.The results indicated that LBP expression in liver was significantly higher than the other tissues(P<0.01).The expression in duodenum was significantly higher than the other tissues except for liver(P<0.05).There were no significant difference among tissues except for liver and duodenum(P>0.05).The expression in 1 and 21 days-old liver were significantly higher than 7,14,28 and 35 days-old(P<0.01),and 158 days-old was significantly higher than 28 and 35 days-old(P<0.01).The expression in 158 days-old liver was higher than 7 and 14 days-old(P<0.05).The LBP expression patterns after various concentrations LPS(0.1 ?g/mL,0.5 ?g/mL and 1 ?g/mL)stimulation were not consistent with those at different development satges.The results speculated that main synthesis of porcine LBP was in liver,and a certain degree LBP expression in intestinal structure indicated intestinal tissues could also secrete and synthetize LBP.The change of external environment would affect expression and the main cell line synthetized porcine LBP was not enterocyte.The potential core promoter region of porcine LBP gene was achieved by using BDGP software.We designed deletion fragments based on predictive analysis results and detected the promoter activity of each fragments by using dual-luciferase reporter system.The results indicated that the LBP promoter(-500?206 bp)was the core promoter region and the fragment(-1500?-500 bp)might be key region regulated promoter activity of LBP.This experiment detected the methylation level of LBP core promoter region by using pyrosequencing.The negative correlation between methylation level CpG2 and CpG3 and LBP expression was significant in different tissues and the linearly dependent coefficient were-0.4684 and-0.3069 respectively.However,there were no significant correlation between methylation and expression in liver at development stages.The methylation of CpG2 and CpG3 might block transcription factors YY1 to bind to correspondent sites resulted in tissue expression difference.Pathogenic bacteria and LPS infecting piglets might affected LBP expression by other regulation approaches. |
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