H1N1亚型流感病毒M1基因重组杆状病毒的构建及鉴定

目的构建H1N1亚型流感病毒M1基因重组杆状病毒,并进行鉴定。方法 PCR扩增H1N1亚型流感病毒(A/PR/8/34)全长M1基因,与pFastBacdua(lpFBD)载体连接,构建重组杆状病毒转移载体pFBD-M1,转化含有Bacimd和Helper质粒的DH10Bac感受态细胞,获得骨架质粒rBacmid-M1,将其转染sf9昆虫细胞,获得重组杆状病毒rBac-M1。采用噬斑形成法检测病毒滴度,PCR法检测M1基因的插入,间接免疫荧光、Western blot和ELISA法检测M1蛋白的表达。结果重组杆状病毒骨架质粒rBacmid-M1经PCR鉴定证实构建正确;第3代rBac-M1病毒滴度为3×108pfu/ml;感染rBac-M1的sf9细胞经PCR扩增可见3 300 bp的条带,间接免疫荧光检测可见特异性绿色荧光,Western blot检测可与鼠抗流感病毒(PR8)和鼠抗M1蛋白(PR8)多克隆抗体发生特异性反应,ELISA检测M1蛋白可与鼠抗流感病毒(PR8)多克隆抗体发生特异性反应,具有良好的反应原性。结论已成功构建了H1N1亚型流感病毒M1基因重组杆状病毒,为进一步研究流感病毒M1的功能及新型流感疫苗开发奠定了基础。

Construction and identification of recombinant baculovirus with M1 gene of influenza H1N1 virus

Objective To construct and identify the recombinant baculovirus with M1 gene of influenza H1N1 virus.Methods Full-length M1 gene of influenza H1N1 virus(A / PR / 8 / 34)was amplified by PCR and inserted into vector pFastBacdual(pFBD).The constructed recombinant baculovirus transfer vector pFBD-M1 was transformed to competent DH10Bac cells containing Bacimd and Helper plasmids.The obtained skeleton plasmid rBacmid-M1 was transfected to sf9 cells,and the constructed recombinant baculovirus rBac-M1 was determined for titer by plaque formation test,for insertion of M1 gene by PCR,and for expression of M1 protein by IFA,Western blot and ELISA.Results PCR proved that skeleton plasmid rBacmid-M1 was constructed correctly.The titer of rBac-M1 of passage 3 was 3 × 108 pfu / ml.The gene band at a length of 3 300 bp was amplified by PCR from,and green fluorescence was observed in the sf9 cells infected with rBac-M1.Western blot showed specific reactions of expressed product with mouse polyclonal antibodies against influenza virus(PR8)and M1 protein(PR8).ELISA showed specific reaction of M1 protein with mouse polyclonal antibody against influenza virus(PR8),indicating high reactogenicity of M1 protein.Conclusion The recombinant baculovirus with M1 gene of influenza H1N1 virus was successfully constructed,which laid a foundation of further study on the function of M1 protein of influenza virus and the development of novel influenza vaccine.