鸭源H7N9亚型流感病毒HA蛋白的真核表达及其免疫保护效力分析
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摘要
为进行鸭源H7N9亚型流感病毒HA蛋白表达纯化及其免疫保护效力分析,本研究以鸭源流感病毒A/Duck/Fujian/S4170/2014 (H7N9)HA基因为模板,PCR扩增后将HA片段克隆至昆虫细胞表达载体p Fast Bac1中。将构建的重组质粒p FBacH7HA转化DH10Bac感受态细胞,提取PCR鉴定为阳性的重组杆粒Bacmid-H7HA,转染SF9昆虫细胞。结果显示重组Bacmid-H7HA表达HA蛋白,并装配成重组杆状病毒,将该病毒感染High Five细胞,大量表达H7-HA蛋白。免疫荧光试验、SDS-PAGE及western blot分析结果表明H7-HA蛋白正确表达且纯度达90%以上,血凝试验表明表达蛋白具有良好的生物活性。利用纯化的H7-HA蛋白免疫SPF鸡后进行攻毒保护试验,评价该蛋白作为免疫原对SPF鸡的免疫保护效力,结果显示HA蛋白免疫组鸡只得到100%保护。以上结果表明本研究表达纯化的H7-HA蛋白可以作为防控H7亚型禽流感的候选亚单位疫苗。
In order to express,purify and evaluate the immune-protective efficacy of the HA protein of a duck-origin H7N9influenza virus strain,the HA gene of the H7N9 avian influenza virus(AIV)strain A/Duck/Fujian/S4170/2014 was amplified by PCR and cloned into the baculovirus expression vector pFastBac1,resulting in the construction of the recombinant pFBacH7HA plasmid.After transforming DH10Bac competent cells,the recombinant Bacmid-H7HA was purified,identified by PCR,and transfected into SF9 insect cells where it was expressed and assembled into recombinant baculovirus.The recombinant baculovirus was used to infect High Five insect cells to express the H7HA protein in large scale.The expressed H7HA protein with a purity of over 90%was validated by indirect immunofluorescence,SDS-PAGE and western blot assay.The hemagglutination assay showed that the purified H7HA protein possessed good biological activity.To evaluate the protection efficacy of the purified H7HA protein as an immunogen,SPF chickens vaccinated with the H7HA protein was challenged with the parental H7N9 virus.It was shown that the vaccinated chickens were 100%protected.These results indicated that the H7HA protein purified in this study could be a promising candidate for developing a subunit vaccine against H7 influenza virus.
In order to express,purify and evaluate the immune-protective efficacy of the HA protein of a duck-origin H7N9influenza virus strain,the HA gene of the H7N9 avian influenza virus(AIV)strain A/Duck/Fujian/S4170/2014 was amplified by PCR and cloned into the baculovirus expression vector pFastBac1,resulting in the construction of the recombinant pFBacH7HA plasmid.After transforming DH10Bac competent cells,the recombinant Bacmid-H7HA was purified,identified by PCR,and transfected into SF9 insect cells where it was expressed and assembled into recombinant baculovirus.The recombinant baculovirus was used to infect High Five insect cells to express the H7HA protein in large scale.The expressed H7HA protein with a purity of over 90%was validated by indirect immunofluorescence,SDS-PAGE and western blot assay.The hemagglutination assay showed that the purified H7HA protein possessed good biological activity.To evaluate the protection efficacy of the purified H7HA protein as an immunogen,SPF chickens vaccinated with the H7HA protein was challenged with the parental H7N9 virus.It was shown that the vaccinated chickens were 100%protected.These results indicated that the H7HA protein purified in this study could be a promising candidate for developing a subunit vaccine against H7 influenza virus.
引文
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[2]Chiapponi C,Faccini S,De Mattia A,et al.Detection of influenza D virus among swine and cattle,Italy[J].Emerg Infect Dis,2016,22(2):352-354.
[3]Nayak D P,Hui E K,Barman S.Assembly and budding of influenza virus[J].Virus Res,2004,106(2):147-165.
[4]Spackman E,Swayne D E.Vaccination of gallinaceous poultry for H5N1 highly pathogenic avian influenza:Current questions and new technology[J].Virus Res,2013,178(1):121-132.
[5]王祥,周东明.基于血凝素(HA)的新型流感疫苗研究进展[J].生命科学,2014,26(09):943-948.
[6]张文俊,薛涛,吴小伟,等.鸭源H5亚型禽流感全禽源分子标记疫苗候选株的构建[J].畜牧兽医学报,2011,45(5):685-691.
[7]张妍,张文俊,李群辉,等.Clade2.3.2 H5N1亚型禽流感疫苗候选株的构建[J].畜牧兽医学报,2012,43(6):915-921.
[8]Deng Lei,Mohan T,Chang T Z,et al.Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses[J].Nat Commun,2018,9(1):359.
[9]Archetti M.Selection on codon usage for error minimization at the protein level[J].J Mol Evol,2004,59(3):400-415.
[10]Song Li,Xiong Dan,Kang Xi-long et al.An avian influenza A(H7N9)virus vaccine candidate based on the fusion protein of hemagglutinin globular head and Salmonella typhimurium flagellin[J].BMC Bio,2015,15:79.
[11]Wu Pei-pei,Lu Ji-hu,Zhang Xue-hua et al.Single dose of consensus hemagglutinin-based virus-like particles vaccine protects chickens against divergent H5 subtype influenza viruses[J].Front Immunol,2017,8:1649.
[12]Abe T,Hemmi H,Miyamoto H,et al.Involvement of the toll like receptor 9 signaling pathway in the induction of innate immunity by baculovirus[J].J Virol,2005,79(5):2847-2858.
[13]Abe T,Matsuura Y.Host innate immune responses induced by baculovirus in mammals[J].Curr Gene Ther,2010,10(3):226-231.