Ⅳ型禽腺病毒Fiber-2(knob)蛋白的表达及免疫效力评价

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 ELD₅₀/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3 269 mg/L。当攻毒剂量为10^4.5 ELD

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

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 10^4.5EID₅₀/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 10^4.5EID₅₀/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 was5×10^3.9ELD₅₀,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.