登革病毒1型和2型国内分离株NSI基因的扩增和克隆

Amplification and Cloning of NS1 Gene of Chinese Local Strains DEN1 and DEN2

登革病毒（Ｄｅｎｇｕｅｖｉｒｕｓｅｓ─ＤＶ）为单链正股ＲＮＡ病毒，其非结构蛋白ＮＳ１在病毒免疫反应中起重要作用。我们在ＮＳ１基因序列设计了一对通用引物，扩增序列长度为４１３ｂＰ。应用逆转录──聚合酶链反应（ＲＴ─ＰＣＲ）成功地扩增了ＤＶ１─４型部分基因片段，采用该引物扩增国内分离株ＤＶ１（ＧＺ）和ＤＶ２（ＨＮ９１）同样出现一条特异扩增带，回收该ＤＮＡ片段，并将ＤＶ１（ＧＺ）和ＤＶ２（ＨＮ９１）ＮＳ１基因部分片段分别克隆到ＰＵＣ１９质粒载体中。经Ｐｓｔ１和ＥｃｏＲ１双酶切分析和通用引物ＰＣＲ鉴定证明重组质粒内含有ＮＳ１基因部分片段。准备进行核甘酸序列分析。同时，以上无性繁殖系的构建，为ＤＶ的核酸杂交研究提供了特异性基因探针。

Dengue viruses have a single-strand plus-sense RNA genome.The NS1 gene can play an important role in the immune reaction of virus,The universal primer set were selected on the NSI gene.A pair of 17-mer oligonucleotides were designed. The amplified products were fragments about 413bp in length.The reverse transcrption and polymerase chain reaction(RT/PCR) were used to amplify gene fragments of DEN1,2,3,4. RNA from chinese local strains DEN1 (GZ)and DENZ (HN91) were also successfully amplifide with universal primer. The DNA fragments of about 413bp were recovered.The DNA fragments of the two local strains were inserted into the plasmid PUC19 respectively. Through screening, two kinds of positive recombinant plasmids were ottaimed. The plasmids were digested with restrictive enzytne and were identifide by PCR.The results showed that the DNA fragments of DNE1 (GZ) and DENZ (HN91) were containd respectively