杆状病毒为载体的重组汉坦病毒S基因在Vero-E6细胞中的表达及其生物活性

摘要

目的 将汉坦病毒HV Z10株S基因(HV5)插入已含CMV基因的杆状病毒转移载体pDual-CMV，筛选重组杆状病毒BAC-pDual-CMV-HVS，转化Vero-E6细胞，用于血清汉坦病毒抗体的检测。方法 以pEGFP-N1质粒为模板，通过PCR扩增CMV基因序列，酶切插入杆状病毒转移载体pFastTBacDUAL,构建含CMV的转移载体pDual-CMV。酶切含HV S基因的pMD19-Z10S质粒，插入pDual-CMV，构建pDual-CMV-HVS，转化感受态DH10BAC，筛选并取重组杆状病毒基因，转染TN细胞，筛选重组杆状病毒BAC-pDual-CMV-HVS。BAC-pDual-CMV-HVS转化Vero-E6细胞，制备抗原片，用于血清中抗汉坦病毒抗体的检测并与传统方法进行比较。结果 经测序成功构建重组杆状病毒转移载体pDual-CMV-HVS，按杆状病毒表达系统操作方法，成功筛选BAC-pDual-CMV-HVS，转化VeroE6细胞，经汉坦病毒抗体阳性血清检测，HV S基因在细胞中得到表达，与传统方法一致。结论 成功筛选BAC-pDual-CMV-HVS重组杆状病毒，可用于汉坦病毒抗原片的制备，该抗原片制备方法简便、无需特殊设备，可用汉坦病毒抗体的检测。%Objective The S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum. MethodsTo generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFPN1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDualCMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells. ResultsThe plasmid pDualCMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum. Conlusion The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.