| 摘要: |
| 为了优化H10亚型禽流感病毒快速检测方法，本研究按照鸡偏嗜性密码子将 A/Jiangxi/IPB13/2013(H10N8)的血凝素（HA）基因序列优化后人工合成，克隆至真核表达载体 pCAGGs。将重组质粒双酶切后测序证明该质粒构建成功。用200 μg的表达载体免疫6周龄SPF鸡，分别在第一次免疫后的30、60天用相同剂量实施第二、三次免疫，末次免疫后的第10天心脏采血，分离血清，制备多克隆抗体。间接免疫荧光试验和western blot 试验证明HA蛋白成功表达，多克隆抗体制备成功。血凝抑制试验结果表明多克隆抗体特异性强，与其他亚型禽流感病毒无交叉反应，抗体效价≥128，该研究为H10亚型流感病毒快速鉴定和研究奠定了基础。 |
| 关键词: 禽流感病毒 H10亚型 多克隆抗体 |
| DOI： |
| 投稿时间：2020-01-03修订日期：2020-03-09 |
| 基金项目:国家自然科学基金(31702262, 31702261)；山东省自然科学基金 (ZR2017BC057, ZR2017BC048)；聊城大学实验技术研究基金资助(26322170257,26322170260) |
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| Preparation?of?Hemagglutinin-specific?Polyclonal Antibody?Against?H10?Subtype?Avian?Influenza?Viruses by DNA Immunization |
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| (College of Agricultural,Liaocheng University,Liaocheng Shandong 252000;China) |
| Abstract: |
| Due to detect H10 subtype avian influenza viruses (AIVs), We synthesized and optimized the HA gene of A/Jiangxi/IPB13/2013(H10N8), and cloned into the mammalian expression vector pCAGGs. The recombinant plasmids were confirmed with double restriction enzyme digestion and DNA sequencing. Then six 6-week-old specific pathogen free chicken was immunized with 200 μg HA plasmid three times at a 30-day interval, respectively. Then the sera were collected at 10 days after the third immunization. Both the results of the indirect immunofluorescence and western blot indicated that the HA genes of recombinant plasmids were successfully expressed, and the HA protein can bind to the antibody in the hemagglutinin-specific polyclonal antibody prepared. Antibody titers detected by hemagglutination inhibition test were greater than 126, and the antisera did not cross-react with AIV of other subtypes and Newcastle disease virus, suggesting high sensitivity and specificity of antisera. To conclude, in this study, the hemagglutinin-specific polyclonal antibody against H10 subtype AIVs was prepared successfully, and it will contribute to identify H10 subtype AIVs quickly and accurately. |
| Key words: Avian?influenza?virus ?H10?subtype ?Hemagglutinin-specific?polyclonal antibody |
