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目的构建共表达H5N1禽流感病毒血凝素(HA)和神经氨酸酶(NA)蛋白的杆状病毒表达系统。方法利用PCR方法分别扩增A/Hubei/1/2010(H5N1)病毒的HA和NA基因,克隆至经改造带有对虾白斑综合病毒(WSSV)早期启动子iel的mpFastBac Dual载体,构建mpFast Bac-HA-NA表达质粒,转化DH10Bac感受态细胞,用获得的Bacmid-HA-NA穿梭质粒转染sf9细胞,获得重组杆状病毒Bac-HA-NA;提取重组杆状病毒Bac-HA-NA的DNA并使用PCR方法检测;利用Western blot检测HA和NA表达,并分别检测重组杆状病毒Bac-HA-NA红细胞凝集能力和神经氨酸酶活性。结果经PCR鉴定,构建的质粒Bacmid-HA-NA正确;Western blot检测表明Bacmid-HANA能在sf9细胞中有效表达HA和NA蛋白,生物活性检测表明Bac-HA-NA能引起红细胞凝集并具有神经氨酸酶活性。结论获得了能同时表达禽流感病毒HA和NA的重组杆状病毒Bac-HA-NA,为进一步研究流感疫苗奠定了基础。
Objective To construct a baculovirus expression system co-expressing the hemagglutinin (HA) and neuraminidase (NA) proteins of H5N1 avian influenza virus. Methods HA and NA genes of A / Hubei / 1/2010 (H5N1) were amplified by polymerase chain reaction (PCR) and cloned into mpFastBac Dual Vector which was transformed with the early promoter of WSSV iel to construct mpFast Bac- HA-NA expression plasmid was transformed into DH10Bac competent cells, and the recombinant baculovirus Bac-HA-NA was obtained by transfecting sf9 cells with the obtained Bacmid-HA-NA shuttle plasmid. The DNA of the recombinant baculovirus Bac-HA-NA The expression of HA and NA was detected by Western blot. The agglutination ability and neuraminidase activity of recombinant baculovirus Bac-HA-NA were detected respectively. Results Bacmid-HA-NA plasmid was identified by PCR. Western blot showed that Bacmid-HANA could effectively express HA and NA protein in sf9 cells. The bioassay showed that Bac-HA-NA can cause agglutination of erythrocytes and nerve Enzyme activity. Conclusions A recombinant baculovirus Bac-HA-NA expressing both HA and NA of avian influenza virus was obtained, which laid the foundation for further research on influenza vaccine.