H9N2亚型AIV NP蛋白的原核表达及其免疫效果  

Immune effect of H9N2 subtype AIV NP protein by prokaryotic expression

作　　者：李小凤 谢芝勋 阮志华 李孟 李丹 张民秀 谢志勤 罗思思 韦悠 谢丽基 曾婷婷 张艳芳 黄娇玲 王盛 LI Xiaofeng;XIE Zhixun;RUAN Zhihua;LI Meng;LI Dan;ZHANG Minxiu;XIE Zhiqin;LUO Sisi;WEI You;XIE Liji;ZENG Tingting;ZHANG Yanfang;HUANG Jiaoling;WANG Sheng(Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China/Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 53000l,China)

机构地区：[1]广西壮族自治区兽医研究所农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室/广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国兽医学报》2024年第6期1113-1119,共7页Chinese Journal of Veterinary Science

基　　金：广西重点研发计划资助项目(桂科AB21076004);桂科专项资助项目(23-2);广西自然科学基金资助项目(2022GXNSFAA035445,2021GXNSFBA196031);广西八桂学者资助项目(2019-A50)。

摘　　要：旨在探讨H9亚型禽流感病毒(avian influenza virus,AIV)NP蛋白对小鼠的免疫效果,为研制AIV疫苗奠定基础。试验将H9N2亚型病毒NP基因的扩增产物连接到pET-32a表达载体中,构建重组质粒pET-32a-NP,并将其转入大肠杆菌BL21(DE3)细胞,IPTG诱导后,通过SDS-PAGE和Western blot验证蛋白表达,用NP重组蛋白免疫小鼠,测定小鼠血清中IgG抗体和脾细胞培养上清多细胞因子分泌情况评价其免疫效果。结果显示:NP基因编码区序列全长1497 bp,NP重组蛋白以可溶性和不可溶性蛋白两种形式存在,Western blot结果可见特异性条带。免疫小鼠后血清产生IgG结合抗体,抗体效价为1∶40000,与对照组相比,IL-2、IL-5、IL-13分泌量极显著提高(P<0.001),IL-6分泌量显著提高。IL-4和IL-12 p70分泌量与对照组相比升高,却没有显著性差异。而免疫组与对照组相比IL-1β、IL-18、GM-CMF、TNF-α、IFN-γ分泌量被抑制,但差异不显著(P>0.05)。结果表明,NP重组蛋白是一种良好的免疫原,为流感疫苗的深入研究奠定了基础。The aim of this study is to investigate the immune effect of H9 subtype avian influenza virus(AIV)NP protein on mice and lay the foundation for the development of avian influenza virus(AIV)vaccine.The H9N2 virus NP gene amplification product was cloned into the pET-32a expression vector,and the protein expression was verified by SDS-PAGE and Western blot,and the immune effect was evaluated by measuring the secretion of supernatant multicytokines in mouse splenocytes culture.The results showed that the total length of the coding region sequence of NP gene was 1497 bp,NP recombinant proteins exist in both soluble and insoluble protein forms,and the specific bands were visible in Western blot.After immunizing mice,serum produces IgG-binding antibodies with antibody titers of 1:40000.Compared with the control group,IL-2,IL-5 and IL-13 were significantly increased(P<0.001),and the secretion of IL-6 was significantly increased compared with the control group.IL-4 and IL-12 p70 secretions were elevated compared with controls,but there was no significant difference.Compared with the control group,the secretions of IL-1β,IL-18,GM-CMF,TNF-αand IFN-γwere inhibited,but the difference was not significant(P>0.05).The results showed that NP recombinant protein is a good immunogen,laying a foundation for in-depth research on influenza vaccine.

关 键 词：H9N2亚型禽流感 NP蛋白 原核表达 细胞因子 

分 类 号：S852.65[农业科学—基础兽医学]