袁小远+杨金兴+张玉霞+孟凯+王友令+艾武

摘要：将新城疫病毒SD强毒株的全基因组cDNA序列进行分段扩增，并将各个片段通过In-Fusion技术连于克隆载体pBR322上，获得了SD毒株的全基因组cDNA克隆。通过分段PCR扩增鉴定及全长测序分析，结果表明，SD毒株的全基因组cDNA克隆构建成功，并且成功引入T7启动子序列，全长基因组序列与亲本毒氨基酸序列的一致性为99.9%。单个序列比对发现，全基因组cDNA克隆中有11处碱基突变，其中只有2处引起氨基酸序列发生改变；整个序列未出现基因的缺失、插入变化，因此全长氨基酸的位数未发生任何变化。SD毒株全长cDNA克隆的成功构建和序列分析为后续的反向遗传操作奠定了关键的技术基础。

关键词：新城疫病毒；基因组全长cDNA；重组质粒；序列分析

中图分类号：S852.65+9.5：Q781 文献标识号：A 文章编号：1001-4942（2017）07-0012-04

Abstract The whole genomic cDNA sequence of SD virulent strain of Newcastle disease virus was amplified in sections， and each fragment was attached to the clone vector pBR322 by In-Fusion technology. The whole genomic cDNA clone was obtained. Through the fragmented-PCR amplification and sequencing analysis， the results showed that the whole genomic cDNA clone of SD virulent strain was successfully constructed and the T7 promoter sequence was successfully imported. The identity of amino acid sequence between the whole genomic cDNA clone and parent strain was 99.9%. Single sequence alignment showed that there were 11 base mutations in the whole genomic cDNA clone， and only 2 sites caused the change of amino acid sequence. However， because of no occurrence of deletion and insertion of genes in the whole sequence， the number of full-length amino acids had no change. The successful construction and sequence analysis of whole genomic cDNA clone of SD virulent strain laid the key technical foundations for future reverse genetic operation.

Keywords Newcastle disease virus； Whole genomic cDNA； Recombinant plasmid； Sequence analysis

新城疫（Newcastle disease，ND）是一种禽类的高度接触性疾病，对全世界养禽业造成巨大经济损失[1，2]。新城疫病毒（Newcastle disease virus，NDV）基因組是单股负链不分节段的RNA，病毒基因组包括六个结构基因，基因组全长为15 186 bp（或15 192 bp）[3]。单独的NDV RNA在细胞内不能够进行转录和翻译蛋白，需要核衣壳蛋白、磷蛋白和大分子蛋白组成核糖核蛋白复合物才能行使转录和翻译的功能[4，5]。

反向遗传操作技术是指将RNA病毒基因组RNA逆转录成cDNA，在体外通过基因突变、基因插入、基因敲除、基因置换等方法人为构建新的具有感染性的病毒，来研究病毒的基因组结构、功能及和病毒宿主之间的相互作用等，也叫全长感染性cDNA克隆技术。这一技术解决了RNA病毒基因组难以操作的难题，同时为新型疫苗的研发提供了新的思路。……