登革2型病毒NGC株NS1基因部分序列pcDNA3.1载体的构建

目的：克隆登革2型病毒NGC株NS1基因部分序列,构建NS1基因部分序列的真核表达载体。方法：用PCR技术扩增登革2型病毒NGC株NS1基因部分序列,并定向克隆入pcDNA3.1（＋）的kpnⅠ、xhoⅠ位点,构建真核重组载体pcDNA3.1（＋）-NS1,转化EcoliDH5a。阳性重组质粒用PCR、酶切及序列测定等方法鉴定。电穿孔法将真核重组载体转染COS-7细胞,RT-PCR鉴定其表达情况。结果：阳性质粒经kpnⅠ及xhoⅠ双酶切及PCR获得了1个核苷酸长度为413 bp的基因,序列测定证实与NGC株NS1基因部分基因序列有99%同源,重组真核质粒转染COS-7细胞经RT-PCR鉴定证实有表达。结论：成功构建了含有登革2型病毒NGC株NS1基因部分序列基因的真核重组载体pcDNA3.1（＋）-NS1。

Construction and Identification of Recombinant Plasmid pcDNA3.1(+) for Eukaryotic Expression of DEN-2 NGC Strain Partial NS1 Gene Sequence

Objective： To clone partial sequence of NS1 gene of DEN-2 virus, strain NGC （NDN） and to construct its eukaryotic expression vector. Methods： NDN sequence was amplified with PCR, and the products were cloned into eukaryotic expression vector pcDNA3.1 （ ＋ ）, then transferred into E. coli DH5a. The positive recombinant plasmids were identified by PCR, restriction enzyme digesting and sequence analysis. The recombinant plasmid was transferred into mammalian COS-7 ceils by electroporation and its mRNA was identified by RT-PCR. Results： Digesting results with restriction enzymes kpn I and xho I , and sequence analysis, showed a fragment of 413 bp was obtained of which the sequence was 99% homology with partial sequence of NDN and pcDNA3.1 （ ＋ ）-NS1 mRNA expressed in COS-7 cells. Conclusion ： The constretion of pcDNA3.1 （ ＋ ） -NS1 is successful, which may facilitate further functional study of NS1 partial gene sequence.