| Swine fever is an acute and severe infectious disease of pigs caused by swine fever virus.CSFV is a member of the Pestivirus genus of the Flaviviridae family and is an enveloped,single-stranded,positive-stranded RNA virus.Because of its high mortality,it has caused huge losses to the worldwide pig industry.Currently,swine fever live vaccine(rabbit source)(also known as swine fever spleen lymphocytic vaccine)and cell vaccine are used to prevent swine fever in China.And the antibody level was commonly used as the immune evaluation method of swine fever vaccine.But it is not comprehensive enough to evaluate the immune effect of swine fever spleen vaccine.Cellular immunity plays important role in the immune effect of swine fever spleen vaccine,but the mechanism still need to be scientifically verified.Therefore,in this study,the transcriptomic technology was used to screen the differentially expressed genes of peripheral blood cells at different times after immunization with swine fever spleen lymphocytic vaccine,and they were validated at the cellular and animal levels.The research results can provide supplementary genes for the induction of cellular immunity by spleen lymphocytic vaccine of swine fever,and are also of great significance for the clinical application of splenic lymphocytic swine fever vaccine.This paper mainly includes three aspects as follows:1.Transcriptomic analysis of peripheral blood cells after immunization of rabbit spleen lymphoid tissue.First,10 4-week-old piglets with double-negative of swine fever antigen and antibody were selected,and divided into two groups equally,physiological saline(group A)and rabbit spleen lymphoid tissue(group B)were injected respectively.Three days later,the peripheral blood of the two groups were collected and put into anticoagulation tubes,then the lymphocytes were isolated and sent to a biological company for transcriptome sequencing.The differentially expressed genes(DEGs)were screened,and the function of EDGs were analyzed by GO and KEGG functional enrichment analysis method.Finally,the total RNAs in two groups were extracted and reverse transcribed into c DNA,s whether The expression levels of DEGs were verified by QPCR.The results showed that the sequencing lengths of group A and B were both about 6×10~9bp.The sequencing quality meets the requirements for further test.A total of 1246 DEGs were screened from the two groups.The GO enrichment results showed that 56 GO terms in biological processes were enriched,which incuded 19 GO terms for cellular composition and 10 GO terms for molecular function.KEGG enrichment results show that a total of 17 significantly enriched KEGG pathways.In this study,we focused on the analysis of the KEGG pathway of T cell receptors and Toll-like receptors.And the expression levels of DEGs were verified by QPCR,results showed that,compared with group A,the expression levels of CD4/8,TCRα,AP-1 and NAFT genes in group B were significantly up-regulated,which was consistent with the transcriptomic data.The above results demostrate that after intramuscular injection three days,the rabbit spleen lymphoid tissue significantly affect the transcriptomic profile of porcine peripheral blood,especially the KEGG pathway that regulates T cell receptors and Toll-like receptors pathway,which improves the immune function of pigs.2.Analysis of the transcriptome profile of peripheral blood cells from swines after immunization with swine fever live vaccine(rabbit source).10 piglets with double-negative of swine fever antigen and antibody at 4weeks old were selected.The swines in group A were injected with swine fever live vaccine(rabbit source)and those in group B were injected with rabbit spleen lymphocytic tissue,respectively.The peripheral blood were anticoagulated and collected at the post injection 3d,6d,and 9d.The lymphocytes were isolated,and were sent to a biological company for transcriptomic sequencing analysis.The DEGs were screened and the function of DEGs were analyzed by GO,KEGG functional enrichment analysis.The total RNAs were extracted from blood samples of each group,and were reverse transcribed to c DNA.Then the expression levels of DEGs were verified by QPCR.The result showed that from the obtained transcriptome data,compared with group A,221 genes were up-regulated and 96 genes were down-regulated in group B at 3d after immunization,596 genes were up-regulated and 332 genes were down-regulated at 6d,and 605genes were up-regulated and 311 genes were down-regulated at 9d.The results of GO enrichment analysis showed that the biological functions of defense response to virus,endometrial system,and microtubule binding were the most significant differences in the three categories of biological process,cellular component and molecular function,respectively.And the results of KEGG analysis showed that the influenza A,epstein-Barr virus infection,RIG-I-like receptor signaling pathway were significantly enriched at the post immune 3d,the influenza A signaling pathway,Epstein-Barr virus infection pathway,Cytosolic DNA-sensing pathway,were significantly enriched at the post immune 6d,and Ribosome,Thermogenesis,AMPK signaling pathway were significantly enriched at the post immune 9d.The results of QPCR verification showed that compared with group A,the expression of CXCL10,RELB,CD86,NOD1,HES4,NQO1,RSP6,OAS1,ISG15 and other genes were up-regulated,and the expression levels of AKT2,MAPK13,STAT1,TRIM25 were down-regulated in group B,which were consistent with transcriptome data.3.HES4 was as a candidate gene for evaluating the immune effect of swine fever spleen vaccine(rabbit source).In this study,DEGs in Notch pathway were analyzed.PK15 cells were treated with swine fever spleen lymphocytic vaccine and rabbit spleen lymphoid tissue for 3 h.Then the mRNAs were extracted and reverse transcribed to c DNA.The expression levels of DEGs were analyzed by QPCR.Then we silent expression of Notch1 by RNAi technology,the expression level of HES4 was determined by QPCR.Finally,30 blood samples were collected from the pig of three farms that were immunized with swine fever spleen vaccine(rabbit source)for 3 days,and the expression level of HES4 was detected by QPCR.The result showed that it was found that the HES4 gene was significantly up-regulated at 3d and 9d after vaccine immunizationby KEGG pathway analysis,,and the QPCR verification results showed that the transcriptome data is accurate.After PK15 cells were treated with spleen lymphocytic vaccine and rabbit spleen lymphocytic inoculation for3d,the mRNA expression level of HES4 was significantly up-regulated in the spleen lymphocytic vaccination group.After silent expression of Notch1 gene,the mRNA expression level of HES4 in the swine fever spleen lymphocytic vaccine group was significantly decreased,too.And the HES4 gene expression was significantly up-regulated in 20 blood samples that were collected from pig farms immunized with swine fever spleen lymphocytic vaccine.It can be seen that the swine fever spleen lymphocytic vaccine can increase the HES4 gene to improve the immune effect of pig through the Notch signaling pathway.In conclusion,immunization of piglets with rabbit spleen lymphoid tissue can significantly affect the porcine peripheral blood transcriptomic profile,especially can regulate the expression levels of DEGs in the T cell receptors and Toll-like receptors KEGG pathway,which improves the immune function of pigs.In addition,compared with immunized rabbit spleen lymphoid tissue,immunization of piglets with live swine fever vaccine(rabbit source)can significantly affect the immune response and inflammation-related pathways.In vitro and in vivo,the live swine,suggesting that HES4 gene can be used as a candidate gene to supplement the cellular immune effect of live swine fever vaccine.The research results will provide the supplement for the evaluation of the immunity effect of live swine fever vaccine(rabbit source). |
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