[抗猪流感病毒H1亚型HA蛋白单克隆抗体的制备]

[Preparation of monoclonal antibodies against HA protein of subtype H1 of swine influenza virus].

作者信息

Li Hong-tao, Liu Ming, Liu Chun-guo, Du Jin-ling

机构信息

National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Mar;25(3):233-5.

PMID:19257989

Abstract

AIM

To prepare the monoclonal antibodies(mAb) against the HA protein of subtype H1 of swine influenza virus (SIV).

METHODS

To construct recombinant expression plasmid subtype H1 of SIV HA gene of A/Swine/Guangdong/LM/2004(H1N1).BALB/c mice of 6-8 weeks old were immunized endemically with the recombinant plasmid.The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells after the last immunization. Hybridoma cells were screened by ELISA and hemagglutination inhibition (HI) tests.The activity and specificity of mAbs were evaluated by HI test and Western blot assay.

RESULTS

Three hybridoma cell lines secreting specific mAbs named 8C4, 8C6, and 9D6 were developed. The ELISA titer of these mAbs was 1:16 000-1:256 000, the HI titer was 1:512-1:256 000; The neutralized titer of 8C4, 8C6, and 9D6 to A/Swine/Guangdong/LM/2004(H1N1) was 10(-2.83), 10(-6.4) and 10(-5.8). Western blot analysis confirmed that mAbs only reacted with HA protein of subtype H1 of SIV.

CONCLUSION

These mAbs can be used as a useful tool to analyze the antigenic variation.They also provide the effective reagents for the rapid detection of subtype H1 of SIV.

摘要

目的

制备抗猪流感病毒（SIV）H1亚型血凝素（HA）蛋白的单克隆抗体（mAb）。

方法

构建A/猪/广东/LM/2004（H1N1）SIV HA基因H1亚型重组表达质粒。用该重组质粒对6 - 8周龄的BALB/c小鼠进行多点免疫。末次免疫后，取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞进行融合。通过酶联免疫吸附测定（ELISA）和血凝抑制（HI）试验筛选杂交瘤细胞。通过HI试验和蛋白质免疫印迹分析评估单克隆抗体的活性和特异性。

结果

获得了3株分泌特异性单克隆抗体的杂交瘤细胞系，分别命名为8C4、8C6和9D6。这些单克隆抗体的ELISA效价为1:16 000 - 1:256 000，HI效价为1:512 - 1:256 000；8C4、8C6和9D6对A/猪/广东/LM/2004（H1N1）的中和效价分别为10（-2.83）、10（-6.4）和10（-5.8）。蛋白质免疫印迹分析证实单克隆抗体仅与SIV H1亚型的HA蛋白反应。