两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究

doi:10.11843/j.issn.0366-6964.2018.07.015

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (7): 1460-1466.doi: 10.11843/j.issn.0366-6964.2018.07.015

两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究

梁文花, 贾云慧, 许程志, 吴运谱, 陈艳, 杨焕良, 乔传玲*, 陈化兰

中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感重点开放实验室, 哈尔滨 150069

收稿日期:2018-01-03 出版日期:2018-07-23 发布日期:2018-07-23
通讯作者: 乔传玲,研究员,主要从事动物流感防控技术的研究,E-mail:qcl@hvri.ac.cn
作者简介:梁文花(1991-),女,山东临沂人,硕士生,主要从事猪流感单克隆抗体研究,E-mail:13011229577@163.com
基金资助:
国家重点研发计划项目（2017YFD0501603）

Preparation and Antiviral Activity of Two Neutralizing Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus

LIANG Wen-hua, JIA Yun-hui, XU Cheng-zhi, WU Yun-pu, CHEN Yan, YANG Huan-liang, QIAO Chuan-ling*, CHEN Hua-lan

Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2018-01-03 Online:2018-07-23 Published:2018-07-23

摘要/Abstract

摘要：

为了制备抗欧亚类禽型（Eurasian avian-like，EA） H1N1猪流感病毒（SIV）血凝素（HA）蛋白的单克隆抗体（MAb）并对其生物学活性进行检测，利用SIV A/swine/Henan/11/2005（HN11）增殖、纯化的全病毒蛋白作为免疫原，免疫BALB/c小鼠，取免疫小鼠的脾细胞与骨髓瘤细胞（SP2/0）融合，采用间接ELISA和HI检测方法进行筛选。结果获得了两株能稳定分泌抗HA蛋白MAb的杂交瘤细胞株，分别命名为2B6和4C7；两株MAbs的腹水ELISA和HI效价均分别高于1：10⁷和5 log2；两株MAbs能够在IFA试验中与病毒HA蛋白发生反应，而在Western blot试验中不能检测到病毒HA蛋白；病毒中和试验结果显示这两株MAbs对EA H1N1 SIV具有病毒中和活性；MAbs的抗病毒活性又进一步通过小鼠的感染试验进行了评估，结果表明接种了MAbs的小鼠获得了有效抵抗同源及异源H1N1 SIV感染的保护效果。研究证实制备的两株MAbs具有较高的病毒中和活性和较强的抗病毒感染能力，可以将其进一步应用于抗EA H1N1 SIV的感染及开展病毒抗原性变异的分子基础研究。

Abstract:

This study aimed to prepare monoclonal antibodies(MAbs) against HA protein of Eurasian avian-like H1N1(EA H1N1) virus and analyze their biological activities. After viral proliferation in chicken embryos, the purified whole-virus protein of A/swine/Henan/11/2005(HN11) was used to immunize BALB/c mice. The splenic cells of the mice were fused with the SP2/0 cells after the third immunization. After screening by indirect ELISA and hemagglutination inhibition test and three circles of sub-clone, two hybridoma cell strains secreting MAbs against HA protein of the HN11 were obtained, which was designated as 2B6 and 4C7, respectively. The ELISA titers of two MAbs were higher than 1:10⁷, and HI titer were higher than 5 log2. These two MAbs could specifically recognize HA protein expressed by plasmid pCAGGS-H1HA in 293T cells by IFA, but not in Western-blot assay. The results of neutralization test indicated that two MAbs have specific neutralization activities for EA H1N1. The protective efficacies of two MAbs were further verified in BALB/c mice, which suggested that MAbs 2B6 and 4C7 can effectively provide protection against the infection with homologous and heterologous H1N1 influenza viruses. Two MAbs, with high viral neutralization activity and strong anti-viral infection ability, were prepared, could be used for further research on anti-viral infection and the molecular basis of antigenic variation of EA H1N1 SIVs.

中图分类号:

S852.659

梁文花, 贾云慧, 许程志, 吴运谱, 陈艳, 杨焕良, 乔传玲, 陈化兰. 两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究[J]. 畜牧兽医学报, 2018, 49(7): 1460-1466.

LIANG Wen-hua, JIA Yun-hui, XU Cheng-zhi, WU Yun-pu, CHEN Yan, YANG Huan-liang, QIAO Chuan-ling, CHEN Hua-lan. Preparation and Antiviral Activity of Two Neutralizing Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1460-1466.

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两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究

Preparation and Antiviral Activity of Two Neutralizing Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus

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