Ⅳ型禽腺病毒Fiber-2(knob)蛋白的表达及免疫效力评价  

Expression of FAdV-4 Fiber-2(knob) protein and evaluation of its immune efficacy

作　　者：程慧敏 刘小晓 林健[1] 刘立新[1] 赵际成 段会娟[1] 王小蕾[1] 范维 刘月焕[1] 钱泓 张强 杨志远[1] CHENG Huimin;LIU Xiaoxiao;LIN Jian;LIU Lixin;ZHAO Jicheng;DUAN Huijuan;WANG Xiaolei;FAN Wei;LIU Yuehuan;QIAN Hong;ZHANG Qiang;YANG Zhiyuan(Institute of Animal Husbandry and Veterinary Medicine,Beijing Municipal Academy of Agricultural and Forestry Sciences,Beijing 100097,China;Zhejiang Hailong Biological Co.,Ltd.,Shaoxing,Zhejiang 312300,China;Zhejiang Sci-Tech University,Hangzhou 310018,China)

机构地区：[1]北京市农林科学院畜牧兽医研究所,北京100097 [2]浙江海隆生物科技有限公司,浙江绍兴312300 [3]浙江理工大学,浙江杭州310018

出　　处：《中国兽医学报》2022年第2期218-222,共5页Chinese Journal of Veterinary Science

基　　金：北京市农林科学院青年基金资助项目(QNJJ202025)。

摘　　要：表达Ⅰ群Ⅳ型禽腺病毒(FAdV-4)Fiber-2(knob)蛋白,以制备Fiber-2(knob)蛋白亚单位疫苗并对其免疫效力进行评价。将FAdV-4 Fiber-2的knob基因克隆至原核表达载体pET28a,构建重组质粒pET28a-Fiber-2(knob)。将重组质粒转化至大肠杆菌BL-21(DE3),以异丙基硫代半乳糖苷(IPTG)诱导重组蛋白表达,采用镍离子亲和层析柱纯化,SDS-PAGE检测纯化后的目的蛋白。将蛋白抗原与佐剂1∶3乳化,分组免疫100,50,25,0 mg/0.5 L FAdV Fiber-2(knob)蛋白亚单位疫苗,攻毒剂量为10^(4.5) ELD_(50)/只,免疫后24 d攻毒,比较不同免疫剂量攻毒保护效果。又制备4批蛋白含量为24 mg/0.5 L FAdV Fiber-2(knob)蛋白亚单位疫苗,比较不同批次间免疫效力差异。结果显示,成功构建pET28a-Fiber-2(knob)重组质粒,纯化后的蛋白纯度达到90%,质量浓度为3269 mg/L。当攻毒剂量为10^(4.5) ELD_(50)时,FAdV Fiber-2(knob)蛋白亚单位疫苗攻毒保护率可达到100%(免疫蛋白抗原量为25~100μg/只),当攻毒剂量为5×10^(3.9) ELD_(50)时,最小免疫蛋白抗原含量为14.4μg/只时也可产生100%保护。不同批次间FAdV Fiber-2(knob)蛋白亚单位疫苗均能刺激机体产生抗体,有效保护SPF鸡抵御FAdV强毒攻击,具有良好的免疫保护效力。The Fiber-2(knob)protein was expressed in groupⅠtypeⅣavian adenovirus(FAdV-4),and the Fiber-2(knob)protein subunit vaccine was prepared and its efficacy was evaluated.The knockout gene of FAdV-4Fiber-2was cloned into prokaryotic expression vector pET28a,yielding pET28a-Fiber-2(knob).The recombinant plasmid was transformed into E.coli BL-21(DE3).The recombinant protein expression was induced by IPTG.The protein was purified by Ni^(2+) ion affinity chromatography column and the purified protein was detected by SDS-PAGE.The protein and adjuvant were emulsified in the ratio of 1∶3.The immune protein content was 100,50,25,0 mg/0.5LFAdV-4Fiber-2(knob)protein subunit vaccine.Twenty-four days after immunization,the chicken was challenged with the virus,the dose of the attack was 10^(4.5)EID_(50)/chicken,and the protective effect of different doses of the vaccine was compared.Four batches of protein subunit vaccine with protein content of 24 mg/0.5L were prepared,and the immune efficacy differences among different batches were compared.The results showed that recombinant plasmid pET28a Fiber-2(knob)was successfully constructed and the purity of purified protein reached 90%,and the mass concentration was 3269mg/L.When the attack dose of the virus was 10^(4.5)EID_(50)/chicken,the protection rate of the FAdV Fiber-2(knob)protein subunit vaccine against FAdV attack was100%(the amount of immunogen antigen was 25-100μg/chicken).When the dose of attack was5×10^(3.9)ELD_(50),the minimum immunogen antigen content of 14.4μg/chicken could also generate100%protection.In conclusion,the FAdV Fiber-2(knob)subunit vaccine can stimulate the body to produce antibodies,effectively protect SPF chickens against the attack of FAdV,and have good immune protection effect.

关 键 词：Ⅳ型禽腺病毒 Fiber-2蛋白 亚单位疫苗 免疫效力 

分 类 号：S852.65[农业科学—基础兽医学]