抗cH5/3抗体Luminex检测系统的构建及其在HA杆部抗体应答研究中的初步应用  

Construction of anti-CH5/3 antibody luminex detection system and its preliminary application in the study of HA stalk antibody response

作　　者：何江 苏敏[1] 刘福慧 张柯 陈瑶 龙兆钇 袁栋波 董俊杰 何张祎檬 黄俊琼 He Jiang;Su Min;Liu Fuhui;Zhang Ke;Chen Yao;Long Zhaoyi;Yuan Dongbo;Dong Junjie;He Zhangyimeng;Huang Junqiong(Department of Blood Transfusion,Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Blood Transfusion,Suining Central Hospital,Suining Sichuan 629200,China;The Department of Medicine of Zunyi Medical University,Zunyi Guizhou 563099,China;School of Traditional Chinese Medicine,Changsha Medical College,Changsha Hunan 410219,China)

机构地区：[1]遵义医科大学附属医院输血科,贵州遵义563099 [2]遂宁市中心医院输血科,四川遂宁629200 [3]遵义医科大学临床医学系,贵州遵义563099 [4]长沙医学院中医学院,湖南长沙410219

出　　处：《遵义医科大学学报》2021年第3期308-313,共6页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:81860376);遵义市科学技术基金项目(NO:遵市社科合社字201887)。

摘　　要：目的构建抗嵌合血凝素cH5/3抗体Luminex检测系统,并初步应用于流感病毒诱导HA杆部抗体研究中对低水平抗HA杆部抗体的检测。方法根据GeneBank中A/VietNam/1203/04(H5N1)流感病毒H5头部和A/Perth/16/2009(H3N2)病毒H3杆部基因序列,人工合成嵌合血凝素cH5/3基因;构建重组Bacmid-cH5/3质粒;重组质粒在sf9细胞中包装成重组cH5/3杆状病毒,并进行PCR及测序鉴定。利用杆状病毒表达系统表达cH5/3蛋白,将纯化蛋白与活化后的微球偶联,构建抗cH5/3抗体Luminex检测系统。分离流感疫苗接种前后健康人外周血B细胞,灭活H7N9病毒刺激6d后,Luminex检测系统分析血清和经H7N9刺激的B细胞培养上清中的抗cH5/3抗体。结果PCR扩增出大小约1700 bp的目的条带,测序鉴定结果证明目的基因序列完全正确。纯化蛋白经Western blot鉴定特异性好;偶联验证实验证实cH5/3蛋白与磁珠偶联成功。Luminx检测系统检测到所有志愿者外周血及灭活H7N9病毒刺激的B细胞培养上清液中均存在抗cH5/3抗体。疫苗接种后的B细胞经灭活H7N9刺激后培养上清中的抗cH5/3抗体水平较疫苗接种前增高4.24(0.8,26.8)倍。结论成功构建抗cH5/3抗体Luminex检测系统,该检测系统检测到H7N9病毒可刺激记忆B细胞产生抗H3杆部抗体,为广谱流感疫苗的研究提供了实验依据。Objective To construct an anti-chimeric hemagglutinin cH5/3 antibody Luminex assay for the detection of low-level anti-HA stalk antibodies in the study of HA stalk-antibody response to influenza viruses.Methods According to the gene sequences of influenza virus H5 head of A/VietNam/1203/04(H5N1)and H3 stalk of A/Perth/16/2009(H3N2)in the GeneBank,the chimeric cH5/3 gene was artificially synthesized,and the recombinant pFastBac1-cH5/3 plasmid was constructed.The recombinant plasmid was identified by PCR and sequencing.The recombinant cH5/3 baculovirus was packaged in SF9 cells.The baculovirus expression system was used to express the chimeric hemagglutinin protein cH5/3.The purified protein was coupled to the activated microspheres to construct an anti-cH5/3 antibody Luminex detection system.Peripheral blood B cells were separated before and after influenza vaccination and stimulated with inactivated H7N9 viruses for 6 days.The anti-H3 stalk antibody levelsin the peripheral bloodand supernatants of B-cell stimulated by inactivated H7N9 were analyzed by the Luminex detection system.Results A band about 1700 bp was detected by PCR,and the sequence of the target gene was correct through sequencing.The purified protein was identified by Western blot with good specificity.The cH5/3 protein was proved to be successfully coupled to the magnetic beads.The anti-H3 stalk antibodies were detected in the peripheral blood and cell supernatants of allvolunteers.B cells were stimulated with inactivated H7N9 for 6 days in vitro,and the fold-change of 4.24(0.8,26.8)in anti-H3 stalk antibodies were found in B cell culture after vaccination when compared to those before vaccination.Conclusion The Luminex detection system for anti-cH5/3 antibodies has been successfully constructed.H7N9 virus can stimulate memory B cells to produce H3 stalk antibodies in vitro.

关 键 词：流感病毒血凝素 杆部 抗体 LUMINEX 应用 

分 类 号：R331.7[医药卫生—人体生理学]