| African swine fever is a highly pathogenic infectious disease of pigs caused by African swine fever virus（African swine fever virus,ASFV）.It is the only known DNA arbovirus.Because of its strong infectivity and lack of effective vaccine,African classical swine fever virus can lead to high mortality of domestic pigs and wild pigs,which has a significant economic impact on the global pig industry.At present,we do not have a thorough understanding of African classical swine fever and there is no safe and effective vaccine production.Therefore,further elucidation of the function of ASFV gene and its immune response mechanism is of great significance for the prevention of ASFV transmission and infection.This study takes p72 protein as the starting point,through sequence analysis,predicts its potential participation in immune response and regulatory elements,and tests the predicted results through experiments to analyze the biological function of p72 protein,deepen our understanding of ASFV,and provide theoretical support and guidance for ASFV detection and vaccine development.During the construction of the prokaryotic expression vector p72,we accidentally obtained a mutant p72?377-428,which deleted 51 amino acids at the amino acid 377-428.The spatial structure predicted by Swiss-Model showed a great change in its three-dimensional conformation.In order to study the function of p72?377-428protein,we expressed p72 and p72?377-428in E.coli system.We use response surface methodology（RSM）based on three-stage Box-Behnken design to optimize protein yield.The results showed that under the optimum conditions（induced at 37℃for 4 hours in the presence of 1m M IPTG）,the expression of p72?377-428protein in E.coli increased stably from 6.1%to 18.6%of the total protein mass.Finally,after three rounds of immunization,the Ig G secretion induced by p72?377-428protein was significantly higher than that of p72 protein.It is worth noting that the mouse antiserum induced by prokaryotic protein specifically recognizes ASFV virions in porcine serum.Our research shows that p72?377-428protein is worthy of further exploration,because it can produce higher protein yield and higher Ig G than p72 protein,which means it has value as a detection reagent and vaccine production.To determine the cellular immune response induced by ASFV p72 protein and its mutants,we measured the levels of Th1 cytokines（IFN-γ）and Th2 cytokines（IL-4）in mice immunized with these two proteins.The results showed that p72 protein showed the ability to induce cellular immunity,and the secretion of IFN-γincreased with the increase of p72 protein stimulation.Surprisingly,the amount of IFN-γproduced by p72?377-428was much lower than that of p72.At the same time,neither p72 nor p72?377-428could detect IL-4.This means that we can explore the relationship between T cell epitopes of p72 by studying the difference of cellular immunity induced by the two proteins.To explore the reasons for the decrease of cellular immunity induced by p72?377-428,according to the results of pre-transcriptional sequencing,we successfully expressed p72and p72?377-428proteins in 293 cells and porcine macrophage cell line 3D4/21 by lentivirus（lentivirus）mediated gene transfer system.QPCR was used to detect the difference of gene expression between them and the control group.It was found that the transcription level of hypoxia inducible factor-1A（HIF1A）increased significantly.Some studies showed that the expression of IFN-γin NK cells increased when HIF1A was knocked out.Based on these results,we preliminarily inferred that p72 protein may affect the expression of IFN-γthrough HIF1A.In summary,we constructed a mutant strain of ASFV p72 protein,p72?377-428,optimized its expression by response surface analysis,and determined the gap between its humoral immunity and cellular immunity ability triggered by p72 protein,and then found that p72 protein affected the expression of cytokine IFN-γthrough the HIF1A pathway.These results can reveal some of the antigenic and immune regulatory mechanisms of the p72 protein,deepen our understanding of the biological properties of the p72 protein,and provide new ideas for us to prevent and control the spread of ASFV and vaccine development. |
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