【摘要】 以鸡胚成纤维细胞培养ＩＢＤＶ弱毒疫苗株Ｄ７８，经蔗糖密度梯度离心纯化，电镜下见典型ＩＢＤＶ粒子。用ＳＤＳ－蛋白酶Ｋ法提取病毒基因组，低熔点胶回收，琼脂糖凝胶电泳，可见ＩＢＤＶ典型双节段基因组。根据已发表的ＩＢＤＶ基因组序列，设计并合成了１对特异扩增ＩＢＤＶＶＰ２基因的引物。以ＩＢＤＶ基因组为模板，利用ＲＴ－ＰＣＲ技术扩增出了１．５ｋｂ的ｃＤＮＡ产物，将ＰＣＲ产物适当处理后与经ＳｍａⅠ酶切的ｐＵＣ１９质粒平端连接，转化大肠杆菌ＤＨ５α，在含Ａｍｐ、Χ－ｇａｌ和ＩＰＴＧ的琼脂平板上培养，产生大量白色菌落。挑取白色菌落，经质粒大小比较分析初步筛选到一重组质粒。以重组质粒ＤＮＡ为模板作ＰＣＲ检测和部分酶切分析证实，该质粒为含Ｄ７８ＩＢＤＶＶＰ２基因的重组ｐＵＣ１９更多还原

【Abstract】 The cloning and identification of VP 2 gene of IBDV is described. The attenuated strain D78 of IBDV was purified by sucrose density gradient centrifugation. After that, typically bisegmented genomic RNA of the virus was extracted and purified. A 18 base pair oligonucleotide primer was designed and synthesized for specific amplification of VP 2 cDNA of IBDV. A 1.5 kb cDNA fragment was amplified by RT PCR with the IBDV genomic RNA as template. Then, the VP 2 cDNA was inserted into pUC19 at SmaⅠ site. The recombinant plasmid was used to transform the competent E.coli cells. A positive clone harboring the IBDV VP 2 cDNA was identified by restriction analysis and PCR using the recombinant pUC19 as template. Cloning and expression of the VP 2 of IBDV in recombinant fowlpox virus vector were still in progress.更多还原