DNA免疫技术制备H10亚型禽流感病毒HA蛋白单因子血清的研究  

Study on Preparation of Hemagglutinin Mono-specific Serum Against H10 Subtype Avian Influenza Viruses by DNA Immunization

作　　者：高鑫鑫 郭晶[1] 薛睿 赵聪慧 李旭勇[1] GAO Xin-xin;GUO Jing;XUE Rui;ZHAO Cong-hui;LI Xu-yong(College of Agricultural,Liaocheng University,Liaocheng,Shandong 252000,China)

机构地区：[1]聊城大学农学院,山东聊城252000

出　　处：《中国兽药杂志》2020年第3期15-20,共6页Chinese Journal of Veterinary Drug

基　　金：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)。

摘　　要：为优化H10亚型禽流感病毒快速检测方法,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200μg的表达载体免疫6周龄SPF鸡,分别在第一次免疫后的第30天和第60天用相同剂量实施第二次和第三次免疫,末次免疫后的第10天心脏采血,分离血清,制备单因子血清。间接免疫荧光试验和western blot试验证明HA蛋白成功表达,单因子血清制备成功。血凝抑制试验结果表明抗体效价≥128,单因子血清特异性强,与其他亚型禽流感病毒无交叉反应。该研究为H10亚型流感病毒快速鉴定和研究奠定了基础。Due to detect H10 subtype avian influenza viruses(AIVs),the HA gene of A/Jiangxi/IPB13/2013(H10N8)was synthesized and optimized,and cloned into the mammalian expression vector pCAGGs.The recombinant plasmids were confirmed with double restriction enzyme digestion and DNA sequencing.Then 6-week-old specific pathogen free chickens were immunized with 200μg 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 128,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,the hemagglutinin-specific polyclonal antibody against H10 subtype AIVs was prepared successfully,and it will contribute to identify H10 subtype AIVs quickly and accurately.

关 键 词：禽流感病毒 H10亚型 单因子血清 DNA免疫 

分 类 号：S852.5[农业科学—基础兽医学]