目的:建立用糖基工程酵母制备流感血凝素的方法,研究其免疫原性,为酵母表达流感疫苗提供基础.方法:通过PCR的方法扩增编码H1N1流感病毒血凝素HA1 (1～330 aa)的基因片段,将HA1基因克隆到表达载体pPIC9质粒上,电转化到糖基工程酵母中,甲醇诱导表达并用镍亲和层析柱纯化重组蛋白,N-糖苷酶F(PNGF)酶切分析N-糖链,Western印迹验证纯化蛋白,免疫小鼠并测定HA1诱导抗体的滴度.结果:获得HA1基因的酵母重组表达菌株,SDS-PAGE分析可见野生型GS115表达的重组HA1相对分子质量约为100×103,而糖基工程酵母GJK01表达的HA1约为60×103,PNGF酶切后相对分子质量均降至45×103左右；经Western印迹检测,这些条带均为目的蛋白条带,野生型和糖基工程酵母表达的HA1分子大小不同是由于不同的N-糖基化修饰引起的.重组HA1免疫小鼠可产生抗HA1抗体,随着抗原剂量的增加,其产生的抗体滴度相应增加；3次免疫后,4μg HA1诱导小鼠产生的抗体滴度最高.结论:利用糖基工程酵母表达制备了低糖化的流感病毒血凝素HA1,该重组蛋白可以诱导小鼠产生HA1抗体,且产生的抗体滴度具有HA1剂量依赖性.%Objective: To prepare hemagglutinin of influenza A virus in glycoengineered Pichia pastoris,and to research their immunogenicities. Methods: The hemagglutinin HA1(1~330 aa) gene fragment of influenza A(H1N1) virus was amplified by PCR and cloned into the expression vector pPIC9. The plasmid pPIC9-Hal was transformed into glycoengineered P.pastoris,the recombinant protein was purified by Ni-affinity chromatography. The purified influenza Hal with/without N-glycosidase F(PNGase F) digestion was identified by Western blotting. After immunized with the recombinant Hal,the anti-Hal antibody in mice serum was detected. Results: The recombi-nant Hal protein could be expressed in yeast after induced with methanol. SDS-PAGE analysis showed that the recombinant HA1 expressed in GS115 was in the size of 100 kD and 60 kD in GJK01. The molecular weight decreased to 45 kD after PNGase F digesting. Western blotting confirmed they were Hal,and there were different TV-glycosylations between the recombinant proteins. Anti-HA 1 antibody was found in serum of Hal immunized mice. After 3rd immunization,the highest titer of anti-Hal was found in 4 (μg HA1 group. Conclusion: The low glycosylated hemagglutinin HA1 protein of influenza A(H1N1) virus was expressed in the glycoengineered P.pichia,it induced anti-Hal antibodies,and the titers of antibody was dose-dependent.
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