巴斯德毕赤酵母多拷贝整合表达组装禽流感病毒样颗粒

【目的】构建多拷贝整合表达禽流感病毒样颗粒的重组巴斯德毕赤酵母,为H5N1亚型禽流感基因工程疫苗提供基础。【方法】以巴斯德毕赤酵母GS115株18S rRNA基因(rDNA)部分序列,插入pPIC9K载体上XbaⅠ和Bsp1407Ⅰ位点间,构建多拷贝整合表达质粒p8K。再以禽流感病毒H5N1毒株基因组RNA为模板,RT-PCR扩增HA、M和NA基因,分别插入多拷贝载体p8K,构建表达质粒 p8K-HA/M/NA。表达质粒分别扩增后线性化,按比例混合,电转化GS115感受态细胞。增菌后,经遗传霉素G418抗性平板筛选、PCR鉴定携带HA、M和NA基因序列的多拷贝整合菌株。阳性菌株增菌、诱导表达、高压均质破碎后,Western-blot检测表达产物。【结果】PCR鉴定阳性的重组菌株,其细胞裂解蛋白,免疫印迹试验可检测到与HA、M₁和NA分子量一致的蛋白条带;电子显微镜可观察到大小约为80~120 nm 的病毒样颗粒,免疫鸡能产生抗禽流感病毒中和抗体。【结论】重组毕赤酵母能够多拷贝整合、表达、组装禽流感病毒样颗粒。

Avian Influenza Virus-like Particles Expressed and Assembled by Multi-copy Recombinant Pichia Pastoris

【Objective】 To construct multi-copy integrative pichia pastoris for expression and assembly of virus-like particles(VLPs) to support the research of genetically engineered vaccine of avian influenza virus(AIV) H5N1subtype. 【Method】 Partial 18S rRNA gene (rDNA) of Pichia pastoris GS115 strain was amplified out and inserted into enzymatic XbaⅠ and Bsp1407Ⅰ sites of plasmid pPIC9K to construct multi-copy integrative vector p8K. And, structural protein HA, M and NA genes of AIV H5N1 subtype were amplified by RT-PCR to construct expression plasmids p8K-HA/M/NA, respectively. Subsequently, those expression plasmids were linearized,, mixed proportionally and electro-transformed into Pichia pastoris GS115 strain, and screened on Geneticin G418 plates. Later, positive recombinants carrying multiple-copied HA, M and NA genes identified by PCR were picked out, enrichment cultured and induction expressed, harvested, broken by High speed homogenation and confirmed by Western-blot, respectively.【Result】 Cell lysates of positive recombinant yeasts confirmed by PCR method c be identified out by immunoblotting with three bands corresponding to HA, M₁ and NA. And, 80~120nm virus-like particles (VLPs) be observed by electron microscopy.chickens injected by VLPs produce specific neutralization antibodies against AIV.【Conclusion】 Recombinant pichia pastoris can multi-integrate, express and assemble AIV VLPs.