论文部分内容阅读
目的构建共表达流感病毒基质蛋白1(matrix protein 1,M1)和基质蛋白2(M2)基因的重组杆状病毒。方法扩增流感病毒A/PR/8/34株的M1和M2全长基因,并将其分别插入到p Fast Bacdual(p FBD)载体的两个多克隆位点,筛选出阳性重组转座载体p FBD-M1/M2,将其转化含有穿梭载体Bacimd的感受态DH10Bac细胞,经同源重组获得重组穿梭载体r Bacmid-M1/M2,在脂质体介导下转染sf9昆虫细胞,包装出重组杆状病毒r Bac-M1/M2。采用噬斑形成法检测重组病毒滴度,PCR法检测重组病毒基因组M1和M2基因插入情况,间接免疫荧光试验(indirect immunofluorescence assay,IFA)和Western blot法检测M1和M2基因的表达。结果经PCR鉴定重组穿梭载体r Bacmid-M1/M2构建正确;第3代r Bac-M1/M2滴度为1×108 pfu/ml;感染r Bac-M1/M2的sf9昆虫细胞经PCR扩增,可见3 600 bp的条带;可在感染细胞的胞内和胞膜上检测到明显的特异性黄绿色荧光;感染r Bac-M1/M2的细胞裂解上清与鼠抗流感病毒(PR8株)多克隆抗体发生特异性反应,在相对分子质量约26 000及11 000处可见特异性反应条带。结论成功构建了共表达H1N1亚型流感病毒M1和M2基因的重组杆状病毒,为构建流感病毒样颗粒以及研制新型广谱流感疫苗奠定了基础。
Objective To construct a recombinant baculovirus coexpressing influenza virus matrix protein 1 (M1) and matrix protein 2 (M2) genes. Methods The full-length M1 and M2 genes of influenza virus A / PR / 8/34 were amplified and inserted into two multi-cloning sites of p Fast Bacdual (p FBD) respectively. The positive recombinant transposon vectors p FBD-M1 / M2 was transformed into competent DH10Bac cells containing shuttle vector Bacimd. The recombinant shuttle vector r Bacmid-M1 / M2 was obtained by homologous recombination and transfected into sf9 insect cells under lipofection. Recombinant baculovirus r Bac-M1 / M2. Recombinant virus titers were detected by plaque formation assay. Insertions of M1 and M2 genes were detected by PCR. Indirect immunofluorescence assay (IFA) and Western blot were used to detect the expression of M1 and M2 genes. Results The recombinant shuttle vector r Bacmid-M1 / M2 was confirmed by PCR. The third generation rBac-M1 / M2 titer was 1 × 108 pfu / ml. The sf9 insect cells infected with rBac-M1 / , A band of 3 600 bp was observed. Significant yellow-green fluorescence was detected on the intracellular and extracellular membrane of infected cells. The cell lysate supernatant infected with r Bac-M1 / M2 and murine anti-influenza virus (PR8 strain ) Polyclonal antibodies specific reaction in the relative molecular mass of about 26 000 and 11 000 visible at the specific reaction bands. Conclusion The recombinant baculovirus coexpressing M1 and M2 genes of H1N1 subtype influenza virus was successfully constructed, which laid the foundation for the construction of influenza virus particles and the development of a new broad-spectrum influenza vaccine.