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目的在毕赤酵母中表达恶性疟原虫膜蛋白Pf332-DBL区。方法采用PCR技术,扩增Pf332-DBL区基因序列,并插入到pPICZαA载体EcoRⅠ和XbaⅠ位点间,电转化至毕赤酵母X-33中,筛选阳性重组酵母,甲醇诱导表达,并通过亲和层析纯化重组蛋白,Western blot进行鉴定。结果获得1株阳性重组酵母;表达的重组蛋白相对分子质量约33000,诱导72h,目的蛋白的表达量最高,占上清蛋白总量的85%;纯化的重组蛋白浓度为800μg/ml,与抗His标签的鼠源单抗和抗Pf332-DBL单抗均可发生特异性反应。结论已成功地在毕赤酵母中表达了恶性疟原虫膜蛋白Pf332-DBL区,为下一步利用该蛋白进行亚单位疫苗的研制及其功能研究奠定了基础。
Objective To express P.falciparum membrane protein Pf332-DBL in Pichia pastoris. Methods The gene sequence of Pf332-DBL was amplified by PCR and inserted into EcoRI and XbaI sites of pPICZαA vector. The recombinant plasmid was electroporated into Pichia pastoris X-33. Positive recombinant yeast was selected and induced by methanol. Purification of recombinant protein by chromatography, Western blot identification. Results A recombinant yeast was obtained. The relative molecular mass of the expressed recombinant protein was about 33000 and induced for 72h. The highest expression level of the target protein accounted for 85% of the total protein. The purified recombinant protein had a concentration of 800μg / ml, His-tag murine monoclonal antibody and anti-Pf332-DBL monoclonal antibody can react specifically. Conclusion The P.falciparum Pf332-DBL region has been successfully expressed in Pichia pastoris, which lays a foundation for the further development of the subunit vaccine and its function study.