H5N1高致病性禽流感病毒HA基因在昆虫细胞中的表达  

The expression of H5N1 highly pathogenic avian influenza virus HA gene in insect cells

作　　者：金秋[1] 刘哲[1] 陆敏华[1] 戴望舒[1] 徐言[1] 张晓[1] 曾晓燕[2] 冯振卿[1] 焦永军[2] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,江苏南京210029 [2]卫生部肠道病原微生物重点实验室江苏省疾病预防控制中心病原微生物研究所,江苏南京210009

出　　处：《南京医科大学学报（自然科学版）》2011年第11期1583-1586,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省自然科学基金(BK2006242;BK2008446;BK2009431);江苏省卫生厅基金(H200857)

摘　　要：目的:扩增高致病性禽流感H5N1亚型病毒血凝素(hemagglutinin,HA)基因,并进行真核表达。方法:抽提毒株A/Jiangsu/08-6基因组RNA,反转录为cDNA,用特异性引物扩增HA基因。HA经BamHⅠ/XhoⅠ双酶切后克隆到pFastBac-GP67B载体上,筛选出阳性重组转座质粒pFastBac-GP67B-HA,并将其进一步转化DH10Bac感受态细胞,通过蓝白斑筛选和PCR鉴定后,获得重组穿梭载体(rBacmid-HA),再通过CellfectinⅡ试剂转染rBacmid-HA至对数生长期的sf9昆虫细胞,进行目的蛋白的真核表达。分别用SDS-PAGE、Western blot、血凝试验以及质谱分析对其进行鉴定。结果:HA基因在sf9细胞表达的蛋白分子量约70 000。血凝试验表明重组HA蛋白可使鸡红细胞发生凝集。质谱分析鉴定该重组蛋白为HA蛋白。结论:重组HA蛋白具有天然HA蛋白的活性,为亚单位疫苗的研制及中和抗体的筛选奠定了基础。Objective： To clone the hemagglutinin（HA） gene from H5N1 highly pathogenic avian influenza virus,and get eukaryotically expression.Methods： Total RNA was extracted from virus A/Jiangsu/08-6（H5N1）,and used to reversely transcribe cDNA.HA gene was amplified with HA gene specific primers and then cloned into the pFastBac-GP67B plasmids with digestion by restriction enzyme BamHⅠ and XhoⅠ.The recombinant plasmid pFastBac-GP67B-HA was identified by restriction enzyme and gene sequencing.Following the transformation of E.coli DH10Bac competent cells with pFastBac-GP67B-HA,the recombinant bacmid rBacmid-HA was identified by blue/white selection and confirmed by PCR analysis.Then rBacmid-HA was transfected into sf9 cells using the Cellfectin Ⅱ reagent to eukaryotically express HA protein.The recombinant HA protein was characterized by SDS-PAGE,Western-blot and hemagglutination test and mass spectrographic analysis.Results： HA protein was efficiently expressed in sf9 cells with a molecular weight of about 70 000.In hemagglutination test,the recombinant protein HA can agglutinate chicken erythrocytes significantly.The mass spectrographic analysis confirmed the protein identity.Conclusion： The recombinant HA protein has the same biological activity as the natural HA protein,and the protein could be used to develop influenza specific subunit vaccine and functional antibody in future research.

关 键 词：H5N1型禽流感病毒 血凝素 杆状病毒昆虫细胞表达系统 血凝试验 

分 类 号：Q786[生物学—分子生物学]