H7N9流感病毒血凝素HA在毕赤酵母中的表达及免疫原性分析  

Immunogenicity of hemagglutinin of H7N9 expressed in yeast Pichia pastoris

作　　者：王莎[1,2] 唱韶红[1] 刘波[1] 巩新[1] 徐威[2] 吴军[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学生命科学与生物制药学院,沈阳110016

出　　处：《军事医学》2015年第6期448-452,共5页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31200082);北京市自然科学基金资助项目(7152112)

摘　　要：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HA^1-525,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HA^1-525,并用糖苷内切酶H（endo H）酶切分析其N-糖链。将制备的HA^1-525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,HA^1-525为均一条带,相对分子质量约58×10^3,与理论大小相当,表明HA^1-525存在甘露糖基化结构。HA^1-525两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 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 p PICZαA. The plasmid p PICZ-HA7-S-was transformed into P. pastoris and the HA^1- 525 was detected by ELISA. Then the HA^1- 525 was precipitated by PEG20000. After immunization with the HA^1- 525,the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition（ HI） test. Results The HA^1- 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 HA^1- 525 decreased to 58 ×10^3after being digested by endo H. Anti-HA7 antibody was found in serum of HA^1- 525 immunized mice and the hemagglutination-inhibition titers reached 1 ∶ 700 after the third doses. Conclusion This study shows the HA^1- 525 expressed in P. pastoris can induce the neutralizing antibody in mice.

关 键 词：H7N9流感病毒 血凝素类 毕赤酵母 红细胞凝集抑制 

分 类 号：R373[医药卫生—病原生物学]