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目的利用毕赤酵母表达制备H7N9流感病毒血凝素[HA 1~525个氨基酸(aa)],并对其免疫原性进行研究。方法经全基因合成获得H7N9流感病毒[A/Hangzhou/1/2013(H7N9)]全长HA,以其为模板,PCR得到片段HA1-525,通过NspⅤ和NotⅠ双酶切连入p PICZαA载体,转入毕赤酵母X33,ELISA筛选阳性克隆。发酵培养后,表达上清经PEG20000沉淀获取HA1-525,并用糖苷内切酶H(endo H)酶切分析其N-糖链。将制备的HA1-525免疫BALB/c小鼠,经ELISA检测特异性HA7抗体滴度,红细胞凝集抑制实验分析血抑活性。结果培养上清用抗HA7抗体经ELISA检测,重组菌成功表达HA7,且Western印迹检测发现有特异性弥散条带,经endo H酶切后,HA1-525为均一条带,相对分子质量约58×103,与理论大小相当,表明HA1-525存在甘露糖基化结构。HA1-525两次免疫小鼠后可产生1∶36 000的抗体滴度。以H7N9裂解苗为抗原,检测其血抑活性为1∶700。结论利用毕赤酵母表达的H7N9流感病毒HA1-525可以诱导小鼠产生针对HA7的中和抗体。
Objective To prepare the H7N9 influenza virus hemagglutinin (HA 1 ~ 525 amino acids (aa)] by using the expression of Pichia pastoris and study its immunogenicity. Methods Full-length HA of H7N9 influenza virus [A / Hangzhou / 1/2013 (H7N9)] was obtained by whole-genome synthesis. The fragment HA1-525 was obtained by PCR and cloned into p PICZαA vector by double digestion with NspⅤ and NotⅠ. Pichia pastoris into X33, ELISA screening positive clones. After fermentation, the supernatant was harvested by PEG20000 precipitation to obtain HA1-525 and its N-glycan was digested with endo H. The prepared BALB / c mice were immunized with HA1-525, and the titer of specific HA7 antibody was detected by ELISA. The hemagglutination inhibition assay was used to analyze the blood anti-tumor activity. Results The culture supernatant was detected by ELISA with anti-HA7 antibody. The recombinant strain successfully expressed HA7, and the specific diffusion band was detected by Western blot. After endo H digestion, HA1-525 was a homogeneous band and the relative molecular mass was about 58 × 103, which is equivalent to the theoretical size, indicating that there is mannosylation structure in HA1-525. Two immunizations with HA1-525 yielded antibody titers of 1: 36,000. H7N9 lysis seedlings as antigens, the test of blood anti-1: 700. Conclusions The use of HA1-525, an H7N9 influenza virus expressed by Pichia pastoris, can induce mice to produce neutralizing antibodies against HA7.