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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。
Objective To construct the H1N1 subtype influenza virus M1 gene recombinant baculovirus and identify it. Methods The full-length M1 gene of H1N1 influenza virus (A / PR / 8/34) was amplified by PCR and ligated with pFastBacdua (lpFBD) vector to construct recombinant baculovirus transfer vector pFBD-M1. The recombinant plasmid was transformed into DH10Bac containing Bacimd and Helper plasmids Competent cells were obtained. The backbone plasmid rBacmid-M1 was obtained and transfected into sf9 insect cells to obtain recombinant baculovirus rBac-M1. The virus titer was detected by plaque formation assay, the insertion of M1 gene by PCR, indirect immunofluorescence, Western blot and ELISA were used to detect the expression of M1 protein. Results The recombinant baculovirus backbone plasmid rBacmid-M1 was confirmed by PCR. The third generation rBac-M1 virus titer was 3 × 108pfu / ml. The sf9 cells infected with rBac-M1 showed a band of 300 bp Specific green fluorescence was detected by indirect immunofluorescence assay. Western blot assay could react specifically with polyclonal antibody of murine anti-influenza virus (PR8) and murine anti-M1 protein (PR8). The detection of M1 protein by ELISA was compatible with murine anti-influenza virus (PR8) polyclonal antibody specific reaction, has good reactionogenicity. Conclusions The recombinant baculovirus of M1 gene of H1N1 subtype influenza virus has been successfully constructed, which lays the foundation for further study on the function of influenza virus M1 and the development of new influenza vaccine.