| In the current swine production,disease,especially infectious is a major problem in pig plagues.Therefore,how to get rid of the excessive dependence on drug to improve pig's immunity and disease resistance is a problem needed to be solved in current industry development.Previous studies have showed that our local pig breeds DPL(Dapulian)is better than foreign pig Landrace in disease resistance.Breeds of DPL and Landrace evolved in different environments,under different pathogen pressure,may be excellent sources of variants of immune response or disease resistance genes.Hence,the study of the immune response and immune-related differentially expressed genes in pig is of great significance to explore the genetic basis and immune mechanism of disease resistance.Oligodeoxynucleotides containing unmethylated CpG motifs(CpG ODN)can interact with Toll-like receptor 9(TLR9)directly activity of innate immune system,inducing to produce Th1-based immune response and indirectly activate the acquired immune system.Therefore,CpG ODN is widely used in the treatment of tumor,cancer,viral infection and allergic diseases.Moreover,CpG ODN has been widely used as vaccine adjuvant.Porcine reproductive and respiratory syndrome(PRRS)was caused by PRRSV.It is characterized by sow reproductive disorders and age-related porcine respiratory disorders.This disease in the world outbreaks frequently,bringing tremendous economic loss to the pig industry.Therefore,an effective inactivated-PRRSV vaccine adjuvant is desiderated to increase the production and safety of inactivated vaccines.1.Comparison of blood physiological and biochemical indexes between DPL and LandraceThe red blood cells,white blood cells,platelets,lymphocytes,intermediate cells,granulocyte results were tested.The results showed that the red blood cells(p<0.01)and lymphocytes(p<0.05)were significantly higher than Landrace pig,and other statistical indexes did not show significant difference(p>0.05).The biochemical indexes showed that the alkaline phosphatase(p<0.05)and albumin(p<0.01)were significantly higher than DPL pig,but the cereal transaminase(p<0.05)was significantly higher than Landrace pig.2.Comparison of immune traits after stimulation DPL and Landrace pig whole blood by CpG ODNThe results showed that the protein expression levels of IL1?(p<0.05)was higher in DPL than Landrace pigs.However,the protein expression levels of IL12(p<0.05)and C reaction protein(p<0.05)were significantly higher in the Landrace pigs.3.Comparison of DEGs after stimulation DPL and Landrace pig by CpG ODNAfter stimulation by CpG ODN,whole blood gene expression profiles of DPL and Landrace pigs were studied using RNA-seq technology.Five Hundred differentially expressed genes(DEGs)were identified between the two breeds.DPL pigs(64.2%,321 gene)had significantly higher number of DEGs than the Landrace(35.8%,179 gene).We performed GO and KEGG pathway enrichment analysis of these DEGs,the results showed that cytokine-cytokine receptor interaction and chemeokine signaling pathway were the major enriched pathways of the immune-relevant DEGs.It was noteworthy that 82.8% of the immune-related DEGs(24/29)were highly expressed in DPL pigs,such as IL1?,IL1?,CXCL8,CXCL10,CCL19,CXCR6(the fold changes of DPL/Landrace were 3.33,1.74,5.63,2.24,5.55 and 3.55).Only,17.2% of the immune-related DEGs were highly expressed in Landrance pigs,such as MyD88,CCL23,CCR2 and IRF5(the fold changes of Landrace/DPL were 6.1845,1.34,1.39 and 1.20).After COG analysis,500 DEGs were classified into 25 COG classes.Among them,the DEGs of R cluster were the most,followed by J and T.Immune-related DEGs screened by RNA-Seq were validated in vivo before and after stimulation.The results showed that the gene expression level of TLR9 was not different.Further more,there was no difference between CXCL9 and IL1? before and after treatment,however,the fold changes of DPL was significant higher than that of Landrance pigs after stimulation.We also found that IL8,CXCL13,CCL3L1 and CCL19 were significantly higher expressed in the stimaltion of DPL pigs,and no significantly differences of IL1? and CXCL2 were found in the two breeds.CXCL10 was significantly higher expressed in the two breeds after stimulation,however,the fold changes of DPL pigs were significantly higher than Landrace pigs after stimulation.After analysis of Single Nucleotide Polymorphism(SNP)and Alternative Splicing(AS),we found that the average number of SNPs of DPL was 81893,and the average SNPs of Landrace was 32021,the DPL piglets had twofold more SNPs and AS than the Landrace pigs.The number of genic SNPs was higher in DPL pigs and the number of Intergenic SNPs was higher in Landrance;while,the Transition,Transversion and Heterozygosity were similar in the two pig breeds.In the SNP type,the base substitutions were higher in DPL than Landrance pigs.SkippedExon,Alt3 splice,IntronRetention and AltFirstExon were the main AS in the two breeds.The AS of SkippedExon and AltFristExon were higher in the DPL pigs.Other AS were similar in the two breeds.After in vitro stimulation PBMCs of DPL and Landrance pigs,the results of qRT-PCR showed that the DPL pigs had significantly higher levels of TLR9 mRNA than those of the Landrance pigs,both before and after CpG ODN stimulation.Cytokine and chemokine induction in the PBMCs of the two breeds were also measured after CpG ODN stimulation.Our data showed that mRNA levels of cytokines(IFN?,IL8,IL12 p40)and chemokines(CXCL9,CXCL13)were significantly higher in the PBMCs of the DPL pigs than those of the Landrace pigs.4.The study of CpG ODN enhances the immune function of PRRS inactivated vaccines.Our results showed that the mRNA levels of Th1 cytokines(IFN-? and IL-12 P40)significantly increased after in the MoDCs incubated with CpG ODN-PRRSV inactivated antigen,and IL4 mRNA of the Th2 cytokines significantly increased.However,only the expression level of IFN-? protein significantly increased.In order to further study the mechanism of action of CpG ODN-PRRSV on MoDCs.We investigated the effect of CpG ODN and PRRSV incubator on TRIF/MyD88-NF-?B signaling pathway.We found that TRIF,MyD88 and NF-?B p65 mRNA expression levels were increased,but only the expression levels of MyD88 and NF-?B P65 were significantly different.At the same time,we also detected the protein expression level of NF-?B P65 in the cells,and the results showed that the protein expression level of the co-incubation group was significantly increased.In summary,the blood physiological and biochemical indexes,the protein expression level of CpG ODN in vitro stimulation whole blood,and the gene expression levels of CpG ODN in vitro and in vitro stimulation in the two breeds were different.In addition,TLR9 ligand CpG ODN enhances the antigen presenting function through the TLR/MyD88-NF-?B signaling pathway.These results provided a theoretical basis for studying the genetic and immunological mechanisms of disease resistance differences between Chinese and foreign pig breeds,and drawn the conclusion that CpG ODN can serve as PRRS vaccine adjuvant. |
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