禽腺病毒血清4型fiber-2和penton蛋白亚单位疫苗初步研究及免疫效果评价  

Preliminary study on fowl adenovirus serotype 4 fiber-2 and penton protein subunit vaccine and evaluation of immune effect

作　　者：刘世佳 尚宵敏 王浩[1] 周睿 李博硕 孙晰瑶 韩静静 杜恩岐[1] LIU Shijia;SHANG Xiaomin;WANG Hao;ZHOU Rui;LI Boshuo;SUN Xiyao;HAN Jingjing;DU Enqi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2023年第6期1162-1166,1186,共6页Chinese Journal of Veterinary Science

基　　金：陕西省国际科技合作与交流计划资助项目(2015KW-027)。

摘　　要：选取禽腺病毒血清4型(fowl adenovirus serotype 4,FAdV-4)JLJ13株的fiber-2和penton蛋白作为保护性抗原,利用SWISS-MODEL网站对fiber-2蛋白空间结构进行模拟,截取其主要功能区fiber-2-Shaft 2替代fiber-2,进行密码子优化后,将fiber-2-Shaft 2和penton蛋白基因序列克隆到pET28a(+)载体上并送至北京天一辉远生物科技有限公司进行基因合成,将合成的质粒分别命名为pET-28a-msyb-fiber-2-Shaft 2和pET-28a-msyb-penton。通过原核表达系统进行蛋白表达并使用镍柱纯化。经BCA定量后,用PBS分别将fiber-2-Shaft 2和penton蛋白质量浓度稀释至1000,500,250 mg/L,再将相同质量浓度的fiber-2-Shaft 2和penton蛋白按1∶1体积比混合后作为抗原,配伍ISCOMs佐剂,0.2 mL/只免疫30日龄SPF鸡,即高剂量组免疫fiber-2-Shaft 2和penton蛋白各100μg、中剂量组各50μg、低剂量组各25μg。一免后14 d进行二次免疫,采集二免后7,14,21 d血清检测特异性抗体和中和抗体,并在二免后21 d攻毒。结果显示,成功表达出fiber-2-Shaft 2和penton蛋白,大小分别为69,93 kDa,镍柱纯化后可获得高纯度的蛋白;二免后14 d高中低量组的中和效价依次为1280,640,640,攻毒后阴性对照组表现出明显的临床症状,2~3 d后死亡,疫苗组均未出现任何临床症状。结果表明,利用禽腺病毒血清4型JLJ13株fiber-2-Shaft 2和penton蛋白混合配伍ISCOMs佐剂制备的亚单位疫苗免疫保护效果明显。To develop a safe and efficient fowl adenovirus serotype 4(FADV-4)subunit vaccine,fiber-2 and Penton proteins of JLJ13 strain of fowl adenovirus serotype 4 were selected as protective antigens in this study.Their gene sequences were searched on NCBI website and fiber-2 protein was spatially-simulated using Swiss-Model website.The main functional area of fiber-2-Shaft 2 was intercepted and cloned into pET28a(+)vector after codon optimization for the gene sequences of fiber-2-Shaft 2 and penton protein.Protein expression was carried out through prokaryotic expression system and purified by nickel column.After BCA quantification,the concentrations of fiber-2-Shaft 2 and penton were diluted to 1000,500,250 mg/L with PBS,respectively.Then,fiber-2-Shaft 2 and penton protein with the same concentration were mixed as antigens according to 1:1 volume ratio,combined with ISCOMs adjuvant.Each dose group was inoculated to SPF chicken of 30 days of age at 0.2 mL.Namely,the high-dose group was immunized with 100μg each of fiber-2-Shaft 2 and penton protein,50μg for medium-dose group and 25μg for low-dose group.The second immunization was performed 14 d after the first immunization.Serum samples were collected at 7,14,21 d after the second immunization and used to detect the specific antibodies and neutralizing antibodies before challenging at 21 d after the second immunization.The results showed that fiber-2-Shaft 2 and penton proteins were successfully expressed and high purity protein was obtained using nickel column purification.The neutralization titers of the high,medium and low dose groups were 1280,640 and 640,respectively.The negative control group showed obvious clinical symptoms after challenge and died 2-3 d later.No clinical symptoms appeared in the immunized group.In summary,the subunit vaccine prepared by the mixture of fiber-2-Shaft 2 and penton protein of avian adenovirus serotype 4 JLJ13 strain with ISCOMs adjuvant has obvious efficacy,which provides a reference for the development of avian adenovirus subun

关 键 词：禽腺病毒血清4型 fiber-2 PENTON 亚单位疫苗 免疫效果评价 

分 类 号：S852.65[农业科学—基础兽医学]