鸡痘病毒NX10株TK基因在病毒复制中不是完全非必需的

发布时间：2018-09-09 19:17

【摘要】：【目的】禽痘病毒(FPV)是痘病毒科禽痘病毒属的成员。FPV因其基因组庞大,含有大量复制非必需区,目前作为活病毒载体在禽类和哺乳动物中广泛使用。重组位点的选择是禽痘病毒载体构建的先决条件,外源基因的插入不影响病毒复制是筛选插入位点的前提。因此,鉴定可供外源基因插入的复制非必需位点将为重组病毒的构建提供更多选择。本研究拟鉴定FPV NX10株胸苷激酶(TK)基因在病毒复制中的必要性。【方法】以TK基因作为靶基因,增强型绿色荧光蛋白(EGFP)为筛选标记构建转移载体,FPV NX10株为亲本病毒,通过同源重组筛选重组病毒r FPV-ΔTK-EGFP。通过在CEF细胞培养物中添加5-溴脱氧尿苷(BUd R)验证TK基因在FPV复制中的作用。【结果】构建了转移载体p UC19-TK AB-EGFP。在转染后重组病毒克隆纯化过程中,蚀斑克隆中绿色荧光病变所占的比例逐渐增加,但在荧光蚀斑的边缘能观察到不带荧光病变的存在。对第9-15轮随机选取的蚀斑克隆的Western blot分析表明,重组病毒中插入的EGFP基因均能够正确表达,但PCR结果显示在重组病毒中始终存在野生型病毒。在细胞培养液中添加BUd R后,重组病毒不能继续生长。【结论】FPV NX10毒株TK基因在该病毒的复制中不是完全非必需的。
[Abstract]:[objective] fowlpox virus (FPV) is a member of the genus of poxvirus. FPV is widely used as a vector of live viruses in poultry and mammals because of its large genome and a large number of non-essential regions for replication. The selection of recombinant sites is a prerequisite for the construction of avian pox virus vectors, and the insertion of foreign genes does not affect the replication of viruses. Therefore, identification of non-essential replication sites for foreign gene insertion will provide more options for the construction of recombinant viruses. The purpose of this study was to identify the necessity of thymidine kinase (TK) gene in viral replication of FPV NX10 strain. [methods] TK gene was used as target gene and enhanced green fluorescent protein (EGFP) was used as screening marker to construct transfer vector NX10 strain as parent virus. Screening recombinant virus r FPV- 螖 TK-EGFP. by homologous recombination The role of TK gene in FPV replication was verified by adding 5-bromodeoxyuridine (5-bromodeoxyuridine) into CEF cell culture. [results] the transfer vector p UC19-TK AB-EGFP. was constructed. In the process of cloning and purification of recombinant virus after transfection, the proportion of green fluorescent lesions in plaque clones increased gradually, but the presence of no fluorescent lesions could be observed at the edge of fluorescent plaque. The Western blot analysis of the randomly selected plaque clones from the 9th to 15th rounds showed that the EGFP gene inserted in the recombinant virus could be expressed correctly, but the PCR results showed that the wild-type virus was always present in the recombinant virus. After the addition of BUd R in cell culture medium, the recombinant virus could not continue to grow. [conclusion] the TK gene of FPV NX10 strain is not completely unnecessary in the replication of the virus.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽呼吸道病创新团队;东北农业大学动物科学技术学院;
【基金】：中国博士后科学基金面上项目(No.2014M560245) 国家科技支撑计划项目(No.2015BAD12B03,2015BAD12B05-05)~~
【分类号】：S852.65

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