禽类多瘤病毒APV-1的cDNA病毒突变体构建

Construction of the cDNA Mutant Recombinant from Avian Polyomavirus 1

为进一步研究禽类多瘤病毒晚期基因多顺反子翻译调控的分子机制,设计和构建了与野生型病毒APV-1相同的在AUG位点有agno-1a突变区和7个插入氨基酸的新型重组病毒.用碱裂解法提取了APV-1 cDNA克隆pHL1003,全长线性目的片段由Acc I部分酶切后回收,再用T4连接酶将合成的agno插入片段磷酸化后与目的片段连接得到了重组质粒,最后转化至E.coli DH10b感受态细胞中筛选阳性克隆.经PCR,PAGE和DNA测序验证获得了APV-1突变cDNA克隆.

To study the molecular mechanism of the poly-cistronic translational control of avian polyomavirus late genes, new recombinants of APV-1 virus with intact agno-la mutant region plus 7 amino acids inserts at its AUG site were designed and constructed. APV-1 cDNA clone pilL1003 was extracted by alkaline lysis method. After retrieving the full linear fragment with Acc I partiM digestion, the synthesized and phosphorylated insert fragment and the retrieved fragment was ligated by T4-1igase, then the recombinant plasmid was transformed to the E. coli DH10b competent cells and the positive clones were screened. PCR, PAGE and DNA sequencing results verified that APV-1 mutant clone was obtained.