Zhu Han-ping, Yao Ping-ping, Wang Jie, Yang Zhang-nv, Xu Fang, Xie Rong-hui, Zhang Yun, Zhu Zhi-yong
Zhejiang Center for Disease Prevention and Control, Hangzhou 310051, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2011 Aug;25(4):280-2.
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.
To generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 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 pDual-CMV, 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.
The plasmid pDual-CMV-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.
[corrected] 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.
将汉坦病毒（HV）Z10株的S基因克隆到含有巨细胞病毒（CMV）启动子的杆状病毒穿梭载体pDual-CMV中，构建重组杆状病毒BAC-pDual-CMV-HVS，然后将重组杆状病毒转染至Vero-E6细胞，应用携带重组杆状病毒的细胞检测HV抗血清。
为构建重组杆状病毒BAC-pDual-CMV-HVS，通过PCR从质粒pEGFP-N1中获取CMV启动子序列，随后克隆到杆状病毒穿梭载体pFastBacDUAL中，得到重组质粒pDual-CMV。然后将HV-S基因序列插入质粒pDual-CMV，构建质粒pDual-CMV-HVS。将质粒pDual-CMV-HVS转化至DH10BAC感受态细胞中，获得重组杆状病毒BAC-pDual-CMV-HVS。将重组病毒转染至Vero-E6细胞，制备抗原底物片。
经测序验证质粒pDual-CMV-HVS构建成功。按照杆状病毒操作流程获得重组病毒BAC-pDual-CMV-HVS，并转染至Vero-E6细胞。用阳性HV抗血清验证了HV-S基因的表达。
成功构建重组病毒并应用于制备抗原底物片。该抗原底物片制备简便，无需特殊设备，可用于检测HV病毒抗血清。
