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.
作者
程慧敏
刘小晓
林健
刘立新
赵际成
段会娟
王小蕾
范维
刘月焕
钱泓
张强
杨志远
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)
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)。
