兼顾H5亚型高致病性禽流感病毒不同分支HA蛋白单克隆抗体的制备及鉴定
|
摘要
为建立针对我国主要流行的H5亚型Clade7.2、Clade 2.3.2和Clade2.3.4.4分支高致病性禽流感病毒(HPAIV)不同流行株的免疫学快速检测方法,本研究将Clade7.2分支禽流感疫苗株CK/LN/S4092/2011(H5N1)(Re-7)、Clade2.3.4.4分支疫苗株DK/GZ/4/2013(H5N1)(Re-8)和Clade2.3.2.1e分支疫苗株DK/AH/S1246/14(H5N1)(Re-10)混合作为免疫原免疫4周龄BALB/c雌鼠,4次免疫后无菌取其脾细胞和骨髓瘤细胞(SP2/0)在聚乙二醇促融剂的作用下融合,通过间接ELISA方法及HI试验筛选获得6株能稳定分泌抗血凝素蛋白(HA)的单克隆抗体(MAb)杂交瘤细胞,分别命名为1B10、2A10、2C8、3E6、4G9和H2B1。经检测1B10、2A10、2C8和H2B1能够与当前AIV流行的分支反应,具有较好的广谱性。本研究为H5亚型AIV快速诊断技术的研发提供基础材料。
To establish a rapid detection method against HA protein of Clade7.2, Clade 2.3.2 and Clade2.3.4.4 H5 subtype avian influenza virus(AIV), which are mainly prevalent in China, the Clade7.2 branch vaccine strain CK/LN/S4092/2011(H5 N1)(Re-7), Clade2.3.4.4 branch vaccine strain DK/GZ/4/2013(H5 N1)(Re-8) and Clade2.3.2.1 e branch vaccine strain DK/AH/S1246/14(H5 N1)(Re-10) were used as an immunogen to immunize four-week old BALB/c female mice. After four immunizations, the splenic cells of mice were fused with SP2/0 cells. Then six hybridoma cell lines which stably secreting the monoclonal antibodies(MAb) against HA protein were screened by indirect ELISA and HI assay, named 1 B10, 2 A10, 2 C8, 3 E6,4 G9 and H2 B1. The MAb of 1 B10, 2 A10, 2 C8 and H2 B1 were able to react with the different recently prevalent H5 Clade AIVs,and they had a broad spectrum. This study provides basic materials for the rapid diagnosis of H5 subtype AIV.
To establish a rapid detection method against HA protein of Clade7.2, Clade 2.3.2 and Clade2.3.4.4 H5 subtype avian influenza virus(AIV), which are mainly prevalent in China, the Clade7.2 branch vaccine strain CK/LN/S4092/2011(H5 N1)(Re-7), Clade2.3.4.4 branch vaccine strain DK/GZ/4/2013(H5 N1)(Re-8) and Clade2.3.2.1 e branch vaccine strain DK/AH/S1246/14(H5 N1)(Re-10) were used as an immunogen to immunize four-week old BALB/c female mice. After four immunizations, the splenic cells of mice were fused with SP2/0 cells. Then six hybridoma cell lines which stably secreting the monoclonal antibodies(MAb) against HA protein were screened by indirect ELISA and HI assay, named 1 B10, 2 A10, 2 C8, 3 E6,4 G9 and H2 B1. The MAb of 1 B10, 2 A10, 2 C8 and H2 B1 were able to react with the different recently prevalent H5 Clade AIVs,and they had a broad spectrum. This study provides basic materials for the rapid diagnosis of H5 subtype AIV.
引文
[1]Yu Zhi-jun,Gao Xiao-long,Wang Tie-cheng,et al.Fatal H5N6avian influenza virus infection in a domestic cat and wild birds in China[J].Sci Rep,2015,5:10704.
[2]WHO(2017).Cumulative number of confirmed human cases for avian influenza A(H5N1)reported to WHO[EB/CD].http://www.who.int/influenza/human_animal_interface/2017-07-25-Table H5N1,2017.
[3]Xu Xi-yan,Kanta S,Cox N J,et al.Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96(H5N1)virus:similarity of its hemagglutinin gene to those of H5N1viruses from the 1997 outbreaks in Hong Kong[J].Virology,1999,261(1):15-19.
[4]WHO/OIE/FAO H5N1 Evolution Working Group.Toward a unified nomenclature system for highly pathogenic avian influenza virus(H5N1)[J].Emerg Infect Dis,2008,14(7):e1.
[5]Sun Hong-lei,Sun Yi-Peng,Pu Juan et al.Comparative virus replication and host innate responses in human cells infected with three prevalent clades(2.3.4,2.3.2,and 7)of highly pathogenic avian influenza H5N1 viruses[J].J Virol,2014,88(1):725-729.
[6]Crowther J R.The ELISA guidebook[M].USA:Humana Press,2009.
[2]WHO(2017).Cumulative number of confirmed human cases for avian influenza A(H5N1)reported to WHO[EB/CD].http://www.who.int/influenza/human_animal_interface/2017-07-25-Table H5N1,2017.
[3]Xu Xi-yan,Kanta S,Cox N J,et al.Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96(H5N1)virus:similarity of its hemagglutinin gene to those of H5N1viruses from the 1997 outbreaks in Hong Kong[J].Virology,1999,261(1):15-19.
[4]WHO/OIE/FAO H5N1 Evolution Working Group.Toward a unified nomenclature system for highly pathogenic avian influenza virus(H5N1)[J].Emerg Infect Dis,2008,14(7):e1.
[5]Sun Hong-lei,Sun Yi-Peng,Pu Juan et al.Comparative virus replication and host innate responses in human cells infected with three prevalent clades(2.3.4,2.3.2,and 7)of highly pathogenic avian influenza H5N1 viruses[J].J Virol,2014,88(1):725-729.
[6]Crowther J R.The ELISA guidebook[M].USA:Humana Press,2009.