H5亚型流感病毒HA头部球状结构域在毕赤酵母中的高效表达及其免疫原性分析

High expression and immunogenicity analysis of HA globular head domain of H5 subtype avian influenza virus produced in Pichia pastoris

摘要:

为了研究H5亚型流感病毒HA蛋白中头部球状结构的免疫原性及其基因优化对蛋白表达的影响，本研究构建了重组真核表达载体pPICZaA-H5HA，并将其转化至毕赤酵母，经筛选获得重组菌株。SDS-PAGE和Western blotting结果分析显示，目的蛋白可在酵母中高效分泌表达，发酵液上清中目的蛋白浓度高达0.2 mg/mL，分子量约为37 kDa。将酵母发酵上清经浓缩、纯化后，获得重组目的蛋白H5HA。将不同剂量的H5HA与不同佐剂联用后分别以滴鼻和肌肉注射两种方式免疫试验动物，进行免疫效力的评价。试验结果表明，H5HA具有较好的免疫原性，可诱导SPF鸡产生较高水平的IgG (血凝抑制效价达1∶64、病毒中和抗体效价为1∶218)，最佳使用剂量为50 μg/羽，与白油佐剂联用时效果最佳，且肌肉注射方式的免疫效果优于滴鼻方式。研究结果为H5亚型流感病毒亚单位疫苗的研制提供了理论支撑。

Abstract:

To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218)?and the optimal?dosage of the recombinant H5HA was 50 μg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.