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目的表达猪流感病毒(swine influenza virus,SIV)H3N2分离株NP基因,获得具有良好抗原性的NP蛋白。方法 PCR扩增猪流感病毒A/swine/Henan/2/2008(H3N2)NP基因,经EcoR I和Xho I双酶切后插入p ET32a(+)载体,并转化大肠杆菌Rosset(DE3)菌株;PCR、EcoR I和Xho I双酶切及DNA测序鉴定重组质粒p ET32a-NP;IPTG诱导,表达、纯化NP并免疫家兔制备免疫血清,SDS-PAGE、WB测其反应原性;同时构建pc DNA3.1-NP并转染293T细胞,免疫荧光检测其免疫源性。结果成功构建重组载体p ET32a-NP和pc DNA3.1-NP,SDS-PAGE和WB显示融合蛋白约80 k D,能与感染SIV的猪血清特异性反应,并且纯化蛋白制备的多克隆血清能识别293T细胞中表达的NP蛋白。结论正确表达SIV H3N2 NP蛋白,并有良好的抗原性,为研究猪流感病毒NP诊断抗原和亚单位疫苗奠定基础。
Objective To express NP gene of swine influenza virus (SIV) H3N2 isolate and obtain NP protein with good antigenicity. Methods The NP gene of swine influenza virus A / swine / Henan / 2/2008 (H3N2) was amplified by PCR and inserted into p ET32a (+) vector after digestion with EcoR I and Xho I. The recombinant plasmid was transformed into Escherichia coli Rosset (DE3) The recombinant plasmid p ET32a-NP was identified by PCR, EcoR I and Xho I digestion and DNA sequencing. IPTG was induced, expressed, purified and immunized rabbits to prepare immune serum. SDS-PAGE and WB were used to measure the reactivity. DNA3.1-NP and transfected 293T cells, immunofluorescence detection of its immunogenicity. Results The recombinant vectors p ET32a-NP and pcDNA3.1-NP were constructed successfully. SDS-PAGE and WB showed that the fusion protein was about 80 kD, and could specifically react with swine sera infected with SIV. The purified polyclonal serum Identify NP proteins expressed in 293T cells. Conclusion The correct expression of SIV H3N2 NP protein with good antigenicity laid the foundation for the study of the diagnostic antigen and subunit vaccine of swine influenza virus.