鸡痘病毒NX10株TK基因在病毒复制中不是完全非必需的

Thymidine kinase gene of Fowlpox virus NX10 strain is partly essential in viral replication

【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。

[Objective] Fowlpox virus（FPV） is a member of Avipoxvirus. FPV is widely used as a living virus vector in poultry and mammals because of its large genome and containing large numbers of replication non-essential regions. The selection of recombination sites is a prerequisite to construct fowlpox virus vectors. The insertion of foreign genes that do not affect viral replication is a prerequisite for selection of the insertion sites. Therefore, identification of replication non-essential regions for foreign gene insertion would provide additional options to construct recombinant viruses. This study aimed to identify the necessity of thymidine kinase（TK） gene in the replication of FPV NX10. [Methods] The recombinant virus r FPV-ΔTK-EGFP was acquired by homologous recombination using FPV NX10 strain as parental virus and enhanced green fluorescent protein（EGFP） as selection marker. The bromodeoxyuridine（BUd R） was added to the culture of CEF cells to identify the role of TK in FPV replication. [Results] The transfer vector p UC19-TK AB-EGFP was constructed. Although the green fluorescent lesions increased in the cloning and purification of recombinant virus, non-fluorescent lesions can still be observed at the edge of the fluorescent plaque. Western blot analysis of the randomly selected plaque clones after 9 to 15 rounds showed that the EGFP gene inserted in the recombinant virus could be expressed correctly, but PCR results showed that the wild-type viruses accompanied with the recombinant virus. After adding BUd R to the cell culture medium, the recombinant virus could not continue to replicate. [Conclusion] The TK gene of FPV NX10 strain is partly essential for replication.