从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质...从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regar...Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.展开更多
文摘从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。
基金supported by the Major Program of National Natural Science Foundation of China (31490603, 31572549)the National Key Technology R & D Program of China (2015BAD12B01-2)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.