【摘要】 本研究团队前期研究发现，猪瘟病毒（CSFV） HCLV株E2蛋白结构域Ⅰ (E2^{Domain Ⅰ})是HCLV株适应家兔的关键结构域，Domain Ⅰ中108位脯氨酸（P）和109位苏氨酸（T）是HCLV株适应家兔必需氨基酸。为进一步探究HCLV适应家兔的分子机制，本研究提取家兔脾脏淋巴细胞膜蛋白分别与CSFV Shimen （SM）株E2蛋白（SM E2）、HCLV株E2蛋白突变体(HCLV E2^P108L-T109I)及HCLV株E2蛋白（HCLV E2）共孵育，经SDS-PAGE后通过蛋白质质谱鉴定，结果却意外发现，CSFV SM E2与家兔脾脏淋巴细胞膜蛋白作用后在37 ku左右处多出一条带；质谱分析结果显示其可能为细胞膜蛋白膜联蛋白1 （ANXA1），且与CSFV SM E2蛋白之间可能存在相互作用。为了分析ANXA1在HCLV适应家兔中的作用，将CSFV SM株和HCLV株分别接种家兔，48 h和72 h后分离各组兔外周血单个核淋巴细胞（PBMC），采用RT-q PCR方法检测ANXA1基因m RNA的转录水平。结果显示，与对照组相比，家兔在接种SM株和HCLV株后，ANXA1基因m RNA转录水平均显著（P<0.05）或者极显著（P<0.01）下调，且与接种HCLV株的家兔相比，接种SM株家兔中ANXA1基因m RNA的转录水平极显著（P<0.001,72 h）或者显著（P<0.05,48 h）下调，表明接种SM株后的家兔可能通过调控ANXA1的转录水平抑制SM株的复制。将pCAGGS-Flag-ANXA1和pCAGGS-Flag分别转染HKE293T细胞，48 h后收集细胞样品，通过GST pulldown验证ANXA1与CSFV SM E2蛋白的相互作用；将pCAGGS-Strep-SME2+pCAGGS-Flag-ANXA1共转染HEK293T细胞作为强毒实验组，将pCAGGS-Myc-HCLV E2+pCAGGS-Flag-ANXA1共转染HEK293T细胞作为弱毒实验组，48 h后采用Co-IP试验验证ANXA1与不同CSFV E2的相互作用。上述结果进一步表明，ANXA1与CSFV SM E2存在相互作用，但其与HCLV E2无相互作用；将pCAGGS-Strep-SME2+pCAGGS-Flag-ANXA1共转染HEK293T细胞作为实验组，采用激光共聚焦显微镜观察发现SM E2蛋白和ANXA1蛋白共定位于细胞浆中。将表达ANXA1的重组质粒转染ST细胞，48 h后分别接种CSFV SM株和HCLV株，采用RT-q PCR检测CSFV基因的拷贝数。结果显示，与转染pCAGGS-Flag的ST细胞相比，接种CSFV SM株的细胞中病毒基因拷贝数极显著上升（P<0.01），而接种HCLV株细胞中病毒基因拷贝数无明显变化。表明过表达ANXA1后可以促进CSFV强毒株的复制，但不影响弱毒株的复制。本研究首次证实，家兔脾脏淋巴细胞ANXA1通过与CSFV SM E2蛋白的相互作用促进CSFV的复制，为进一步研究CSFV E2蛋白的功能奠定了基础。更多还原

【Abstract】 Previous study demonstrated that Domain Ⅰ on the E2 glycoprotein (E2^{Domain Ⅰ}) and P108 and T109 in the domain Ⅰ are critical for the adaptation of swine fever rabbit attenuated vaccine strain （HCLV） to rabbits.To investigate the adaptation mechanism of HCLV strain to rabbits,this study utilized protein pulldown technique to analyze the interaction between the membrane proteins from rabbit splenic lymphocytes with the CSFV Shimen （SM） strain E2 protein （SM E2）,HCLV strain E2mutant protein (HCLV E2^{P108L-T109Ⅰ}),and HCLV strain E2 protein （HCLV E2）.After SDS-PAGE separation,the protein samples were identified by mass spectrometry.Unexpectedly,the results showed that after the interaction between CSFV SM E2 and rabbit splenic lymphocyte membrane proteins,an additional band appeared around 37ku,and mass spectrometry analysis suggested that it might be membrane-associated protein annexin A1 （ANXA1） potentially and interacts with CSFV SM E2 protein.To analyze the role of ANXA1 in HCLV adaptation to rabbits,rabbits were separately inoculated with CSFV SM and HCLV,and peripheral blood mononuclear cell （PBMC） were isolated from each group of rabbits at 48 hours and 72 hours post-inoculation.The transcription levels of ANXA1 m RNA were detected using the RT-q PCR method.The results showed that compared to the control group,the transcription level of ANXA1 m RNA was significantly （P<0.05） or extremely significantly （P<0.01） downregulated in rabbits inoculated with SM and HCLV strains.Moreover,the transcription level of ANXA1 m RNA in rabbits inoculated with the SM strain was significantly （P<0.001,72 hours） or extremely significantly （P<0.05,48 hours） decreased compared to that in rabbits inoculated with the HCLV strain,indicating that rabbits inoculated with the SM strain may suppress SM replication through the regulation of ANXA1 transcription.HKE293T cells were transfected with pCAGGS-Flag-ANXA1 and pCAGGS-Flag respectively,and cell samples were collected 48 hours later for GST pulldown assay to verify the interaction between ANXA1 and CSFV SM E2 protein.HEK293T cells were co-transfected with pCAGGS-Strep-SME2 plus pCAGGS-Flag-ANXA1 as the high virulence experimental group,and pCAGGS-Myc-HCLVE2 plus pCAGGS-Flag-ANXA1 as the low virulence experimental group.After 48 hours,Co-IP assay was performed to validate the interaction between ANXA1 and different CSFV E2 proteins.The above results further demonstrated that ANXA1 interacted with CSFV SM E2,while it had no interaction with HCLV E2.HEK293T cells were co-transfected with pCAGGS-Strep-SME2 and pCAGGS-Flag-ANXA1,and SM E2 protein and ANXA1 protein were co-localized in the cytoplasm by laser confocal microscope observation.after overexpressing ANXA1 in ST cells,CSFV SM and HCLV strains were separately inoculated,and the copy number of CSFV genes was detected by RT-qPCR.Compared with the pCAGGS-Flag-transfected ST cells,a significant increase （P<0.01） in the viral gene copy number in cells inoculated with CSFV SM strain,while no significant change was observed in cells inoculated with HCLV strain.This indicates that overexpression of ANXA1 can promote the replication of virulent CSFV strains,but does not affect the replication of low virulent strains.This study provides the first evidence that ANXA1 promotes the replication of CSFV through the interaction with CSFV SM E2 protein,which lays the foundation for further investigation into the function of CSFV E2 protein.更多还原