SARS病毒S2蛋白在毕赤酵母中的表达及鉴定

目的研究用毕赤酵母菌表达SARS病毒S2蛋白亚单位。方法依据GenBank中SARS基因组序列,采用人工合成的方法合成SARS病毒刺突蛋白S2亚单位的基因(1590bp),再与CTL表位基因(195bp)重组后,将其克隆到毕赤酵母表达载体pPIC9K中,构建重组表达质粒pPIC9K-S2,转化毕赤酵母GS115,并经MD和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115/pPIC9K-S2。经诱导、表达,并对表达产物进行分析、鉴定。结果所构建的重组质粒pPIC9K-S2正确,在毕赤酵母中可高效表达,其表达产物与阳性SARS血清产生特异性反应。结论SARS病毒刺突蛋白S2亚单位可在毕赤酵母中高效表达,产物具有良好的抗原特异性。

Expression of Severe Acute Respiratory Syndrome(SARS) Virus S2 Protein in Pichia pastoris

Objective To express severe acute respiratory syndrome(SARS) virus S2 protein in Pichia pastoris.Methods A 1 590 bp gene fragment encoding the S2 protein of SARS virus was synthesized according to the SARS virus genome sequence reported in GenBank and recombined with CTL epitope gene(195 bp),then cloned into Pichia pastoris expression vector pPIC9K.The constructed recombinant plasmid pPIC9K-S2 was transformed to Pichia pastoris GS115,and the positive clones were screened by MD and MM plate cultures and identified by PCR.The obtained recombinant Pichia pastoris strain GS115/pPIC9K-S2 was induced with methanol,and the expressed product was identified by SDS-PAGE and Western blot.Results Recombinant plasmid pPIC9K-S2 was correctly constructed and highly expressed in Pichia pastoris.The expressed product showed specific reaction with SARS virus-positive serum.Conclusion SARS virus S2 protein was successfully expressed in Pichia pastoris and showed good antigenic specificity.