共表达流感病毒基质蛋白1和基质蛋白2基因的重组杆状病毒的构建  

Construction of recombinant baculovirus for co-expression of M1 and M2 genes of influenza virus

作　　者：姚立红[1] 陈爱珺[1] 徐鹏卫[1] 张智清[1] 郭建强[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京100052

出　　处：《中国生物制品学杂志》2015年第1期1-5,共5页Chinese Journal of Biologicals

摘　　要：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 recombinant baculovirus for co-expression of matrix protein 1 （M1） and matrix protein 2 （M2） genes of influenza virus. Methods Full lengths of M1 and M2 genes of influenza H1N1 virus （A/PR/8/34） were amplified by PCR separately and inserted into the two polyclonal sites of vector pFastBacdual （pFBD） successively. Positive recombinant transposition vector pFBD-M1/M2 was transformed to competent DH10Bac cells containing shuttle vector Bacimd, based on which recombinant shuttle vector rBacmid-M1 / M2 was obtained by homologous recombination, and transfected to sf9 cells in mediation of liposome for packaging. The obtained recombinant baculovirus rBac-M1 / M2 was determined for titer by plaque formation test, for M1 and M2 genes in genome by PCR, and for expressions of M1 and M2 genes by indirect imunofluorescence assay （IFA） and Western blot. Results PCR proved that recombinant shuttle plasmid rBacmid-M1/M2 was constructed correctly. The titer of rBac-M1/M2 of passage 3 was 1 × 10^8 pfu/ml. A band at length of 3 600 bp was amplified from sf9 cells infected with rBac-M1/M2 by PCR. Obviously specific yellowish-green fluorescence was observed in the infected cells. The lytic supernatant of cells infected with rBac-M1/M2 showed specific reaction bands, with relative molecular masses of about 26 000 and 11 000 respectively, showed specific reaction with mouse polyclonal antibody against influenza virus PR8 strain. Conclusion Recombinant baculovirus for co-expression of M1 and M2 genes of influenza H1N1 virus was construct successfully, which laid a foundation of construction of influenza virus-like particles and development of novel broad vaccine against influenza.

关 键 词：流感病毒 基质蛋白1 基质蛋白2 杆状病毒 

分 类 号：R373.13[医药卫生—病原生物学] Q78[医药卫生—基础医学]