CAJ | 학술논문

目的构建含有H1N1亚型流感病毒NA基因的重组杆状病毒.方法用PCR方法扩增甲型H1N1亚型流感病毒(A/PR8/34)全长神经氨酸酶基因,并将其连接于pFastBacdual载体,构建杆状病毒转移载体pFBD-NA.转化含有Bacmid的DH10B感受态细胞后,获得重组穿梭载体rBacmid-NA.将其转染昆虫细胞sf9,获得重组病毒rBac-NA.提取重组杆状病毒rBac-NA的DNA并使用PCR方法检测；采用间接免疫荧光,SDS-PAGE,Western Blot鉴定以及ELISA方法检测所表达的NA蛋白.结果经PCR鉴定所构建质粒rBacmid-NA正确；使用间接免疫荧光检测可见特异性绿色荧光位于感染细胞的表面,Western Bolt检测可与小鼠抗PR8株多克隆抗体和兔抗甲型流感病毒NA多克隆抗体发生特异性免疫反应；经ELISA检测,NA蛋白可被鼠抗流感病毒(PR8)多克隆抗体特异性识别.结论上述结果证明,我们获得了表达流感病毒NA蛋白的重组杆状病毒,为进一步研究NA蛋白的功能和新型流感疫苗的开发奠定了基础.
목적 구건함유H1N1아형류감병독NA기인적중조간상병독.방법 용PCR방법확증갑형H1N1아형류감병독(A/PR8/34)전장신경안산매기인,병장기련접우pFastBacdual재체,구건간상병독전이재체pFBD-NA.전화함유Bacmid적DH10B감수태세포후,획득중조천사재체rBacmid-NA.장기전염곤충세포sf9,획득중조병독rBac-NA.제취중조간상병독rBac-NA적DNA병사용PCR방법검측；채용간접면역형광,SDS-PAGE,Western Blot감정이급ELISA방법검측소표체적NA단백.결과 경PCR감정소구건질립rBacmid-NA정학；사용간접면역형광검측가견특이성록색형광위우감염세포적표면,Western Bolt검측가여소서항PR8주다극륭항체화토항갑형류감병독NA다극륭항체발생특이성면역반응；경ELISA검측,NA단백가피서항류감병독(PR8)다극륭항체특이성식별.결론 상술결과증명,아문획득료표체류감병독NA단백적중조간상병독,위진일보연구NA단백적공능화신형류감역묘적개발전정료기출.
Objective To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.Methods Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA.Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids.After transfecting into sf9 cells,recombinant baculovirus rBac-NA was obtained.The rBac-NA genome was extracted and identified by PCR.NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA,Western Bolt and ELISA.Results PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed.NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells.NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot.ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8),indicating high antigenicity.Conclusion Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed.This work provides a basis for further study on NA protein function and novel influenza vaccine development.