牛副流感病毒3型核衣壳蛋白基因的真核表达

Eukaryotic expression of nucleocapsid protein gene from bovine parainfluenza virus type 3

根据牛副流感病毒3型(BPIV-3)内蒙09株(NM09)全基因组序列(GenBank登录号:JQ063064)设计特异性引物,利用RT-PCR方法扩增出牛副流感病毒3型分离株的核衣壳蛋白(N)基因,通过NheI和NotI限制性内切酶位点亚克隆至真核表达载体pcDNA3.1/Zeo(+),获得真核重组质粒pcDNA3.1-N。采用Superfect转染试剂将重组质粒转染至BSR细胞中,转染后的BSR细胞经间接免疫荧光试验和RT-PCR方法检测,重组质粒pcDNA3.1-N在BSR细胞中能正确表达N蛋白。本研究结果为BPIV-3新型疫苗的研制奠定基础。

The aim of this study is to construct an eukaryotic expression plasmid for nucleocapsid protein （N） gene of bovine parainfluenza virus type 3 （ BPIV-3 ）. According to the complete sequence of BPIV-3 NM09 strain （ GenBank accession ： JQ063064 ）, a pair of primers was designed and synthesized. The N gene of BPIV3 was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The N gene was cloned into pcDNA3. 1 （＋） vector using NheI and NotI, and thus the expression plasmid pcDNA3.1-N was successfully construc- ted. Then pcDNA3. 1-N was transfected into BSR cell using lipoplast mediate method and positive results were demonstrated by indirect im- munofluorescence assay （IFA） and RT-PCR. The results showed N gene of BPIV-3 was successfully expressed in BSR cell. The study pro- vided fundamental information for the further study on the prevention of BPIV3 infection and new vaccine development.