Abstract: In order to prepare Newcastle disease virus HN protein and study its immunogenicity,the optimized extracellular domain of HN gene was cloned into pPICZαA,and the pPICZαA-HN was electroporated into Pichia pastoris X-33 competent cell.Positive transformants were obtained by PCR identification,and induced by methanol, and the strain expressing HN protein was screened by SDS-PAGE and Western blot analysis. The optimal expression conditions were explored by optimizing the methanol content,induction time,initial pH value of the medium,and induction temperature.The target protein was purified by nickel column affinity chromatography and identified by SDS-PAGE,Western blot and indirect ELISA.The purified protein was treated with the deglycosylation enzyme PNGase F to verify the degree of glycosylation.The results showed that the HN recombinant protein was successfully expressed in Pichia pastoris,and the optimal expression condition of HN protein was at 28℃ ,the initial pH of the medium was 7.0,after induction with 0.5% methanol for 5 days.Purified protein was obtained by nickel column affinity chromatography and its purity reached 90%.ELISA showed that the recombinant protein had good activity.Deglycosylation assays demonstrated the presence of glycosylation modifications in recombinant proteins.In summary,this study successfully expressed HN protein using Pichia pastoris X-33,and the purified protein had high purity,good activity and glycosylation modification.

Key words: Newcastle disease virus, HN protein, Pichia pastoris; Protein purification, Protein activity

摘要： 为了制备新城疫病毒（NDV）HN蛋白并研究其免疫原性，将优化的HN基因胞外区插入毕赤酵母表达载体pPICZαA中，构建毕赤酵母表达载体pPICZαA-HN，并将其电转入毕赤酵母X-33感受态中，经PCR鉴定得到阳性转化子，甲醇诱导表达后，经SDS-PAGE和Western blot鉴定筛选出目的蛋白表达菌株；通过对诱导剂含量、诱导时间、培养基初始pH值和诱导温度进行优化，研究最佳表达条件；采用镍柱亲和层析对目的蛋白进行纯化，并用SDS-PAGE、Western blot和间接ELISA对目的蛋白进行鉴定；利用去糖基化酶PNGase F对目的蛋白进行处理，验证其糖基化修饰程度。结果显示，HN重组蛋白在毕赤酵母中成功表达，且在28 ℃条件下，培养基初始pH值为7.0，用0.5%甲醇诱导5 d，蛋白质表达量最高；通过镍柱亲和层析可获得纯度高于90%的重组蛋白；间接ELISA结果表明，重组蛋白活性良好；去糖基化试验验证了重组蛋白存在糖基化修饰。综上，利用毕赤酵母X-33成功表达了HN蛋白，且纯化产物纯度较高、活性良好，具有糖基化修饰。

关键词: 新城疫病毒, HN蛋白, 毕赤酵母, 蛋白质纯化, 蛋白质活性