马抗H7N9亚型流感病毒精制免疫球蛋白的制备及其治疗效果评价

Preparation and curative effect evaluation of equine anti-influenza H7N9 virus immunoglobulin

目的制备马抗H7N9亚型流感病毒精制免疫球蛋白,并评价其对BALB/c小鼠的治疗效果。方法应用昆虫细胞-杆状病毒表达系统分别构建的表达H7N9亚型流感病毒HA、NA及M1蛋白的病毒样颗粒制备抗原,免疫健康马匹,当抗体效价达1∶10 000以上时,采血,对血清进行粗处理;采用亲和层析的方法获得纯化的马抗H7N9免疫球蛋白F(ab’)_2。对纯化的免疫球蛋白进行蛋白含量测定和质量检测。用H7N9亚型流感病毒BALB/c小鼠感染模型评价F(ab’)_2的治疗效果。结果免疫马匹血清中产生高滴度的中和抗体,获得的纯化马抗H7N9免疫球蛋白F(ab’)_2中和抗体效价达1∶5 120,质量检测合格。攻毒4 h后给药0.4和0.2 mg,小鼠存活率均达100%。结论获得了安全、高效的马抗H7N9亚型流感病毒精制免疫球蛋白F(ab’)_2,为人禽流感治疗用制剂的研发提供了参考。

Objective To prepare equine anti-influenza H7N9 virus immunoglobulin and evaluate its curative effect in BALB / c mice.Methods The virus-like particles（VLPs）based on A / Shanghai / 2013 and comprising hemagglutinin（HA）,neuraminidase（NA）,and matrix（M1）were produced using a insect cells-baculovirus expression system,and used for vaccination of horses.The horses were bled when the titer of antibody titer was more than 1 ∶ 10 000,and the sera were treated preliminarily.The F（ab＇）_2of anti-influenza H7N9 virus Ig G was purified by affinity chromatography,determined for protein content and subjected to control tests,of which the curative effect was evaluated in BALB / c mouse model of influenza H7N9 virus infection.Results High titer neutralizing antibody was detected in the sera of immunized horses.The purified F（ab＇）_2against influenza H7N9 virus reached a neutralizing antibody titer of more than 1 ∶ 5 120,and was qualified in control tests.Both the survival rates of mice immunized with 0.4 and 0.2 mg of F（ab＇）_2 4 h after virus challenge were 100%.Conclusion A safe and effective equine F（ab＇）_2 of Ig G against influenza H7N9 virus was obtained,which provided a reference for development of preparations for treatment of avian influenza in humans.