H7 N9流感病毒血凝素HA在毕赤酵母中的表达及免疫原性分析

摘要

Objective To express the hemagglutinin of H7N9 in Pichia pastoris and analyze its immunogenicity. Methods The HA [1 -525 amino acids(aa)] of H7N9 [A/Hangzhou/1/2013(H7N9)] lacking the C-terminal transmembrane anchor-coding sequence was amplified by PCR and cloned into the expression vector pPICZαA.The plasmid pPICZ-HA7-S-was transformed into P.pastoris and the HA1-525 was detected by ELISA.Then the HA1-525 was precipitated by PEG20000.After immunization with the HA1-525 , the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition ( HI) test.Results The HA1-525 was expressed in P.pastoris after being induced with methanol. Western blotting confirmed that there were specific dispersion bands and the molecular weight of HA1-525 decreased to 58 × 103 after being digested by endo H.Anti-HA7 antibody was found in serum of HA1-525 immunized mice and the hemaggluti-nation-inhibition titers reached 1∶700 after the third doses.Conclusion This study shows the HA1-525 expressed in P.pastoris can induce the neutralizing antibody in mice.%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pPICZαA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∶36000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。