传染性喉气管炎病毒中国王岗株tk基因的克隆及鉴定

Cloning and Identification of Thymidine Kinase Gene from a Virulent Chinese Field Isolate of Infectious Lary- gotracheitis Virus

以质粒pBluescript SK^+为载体克隆传染性喉气管炎病毒(ILTV）中国王岗株的DNA,构建了THind Ⅲ DNA文库。参考ILTV美国632株胸苷激酶（tk）基因的DNA序列,设计并合成了一对20bp长的引物;以ILTV中国王岗株DNA为模板,用PCR方法特异性地扩增出ILTV-713bp的tk DNA片段。用光敏生物素标记该713bp片段作为探针,通过斑点杂交从ILTV中国王岗株Hind Ⅲ DNA文库中筛选出4个含2.4kb外源片段的tk基因阳性重组质粒。经酶切分析、Southern杂交、PCR检测和部分限制酶酶谱分析表明:ILTV中国王岗株DNA 2.4 kb Hind Ⅲ 片段无论是片段大小还是酶切图谱均与美国632株完全相同,而且Southern杂交和PCR检测均为tk阳性,证明其中包含有完整的tk基因。本研究的目标是用克隆的tk基因进行体外缺失和重组,以构建毒力进一步降低的tk缺失减毒株,这些工作正在进行中。

To localize the gene encoding the thymidine kinase(tk) gene from a virulent Chinese field isolate of ILTV, a library of the Chinese ILTV strain genome was constructed in the pBluescript SK+ Vector. A pair of primers from the sequence data of tk gene in the virulent US isolate of ILTV were used in the PCR to amplify a 713 base pair(bp) tk fragment of DNA from Chinese strain of ILTV. The 713 bp DNA fragment was used in dot-blot and Southern blot to screen the recombinant plasmid of the tk from the Chinese ILTV strain and to localize the ILTV tk gene on a 2. 4 kb Hind III fragment of the Chinese ILTV strain. It was domonstrated that the restrict map of tk gene from the virulent US isolate of ILTV was in accord with the restrict map of the tk gene from the virulent Chinese isolate of ILTV.