恶性疟原虫膜蛋白Pf332-DBL区在毕赤酵母中的表达

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。

Expression of Pf332-DBL Region of Plasmodium falciparum Membrane Protein in Pichia pastoris

Objective To express the Pf332-DBL region of Plasmodium falciparum membrane protein in Pichia pastoris.Methods The gene sequence encoding Pf332-DBL region was amplified by PCR and inserted into vector pPICZαA,between the restriction sites of EcoRⅠand XbaⅠ,then transformed to Pichia pastoris X-33 by electrotransformation.The positive recombinants were screened for expression under induction of methanol.The expressed product was purified by affinity chromatography and identified by Western blot.Results One positive recombinant was screened,and the relative molecular mass of expressed protein was 33 000.The expression level of target protein reached a peak value 72 h after induction,accounting for 85%of total protein in supernatant.The purified recombinant protein reached a concentration of 800μg/ml and showed specific reactions with murine McAb with anti-His label and the McAb against Pf332-DBL.Conclusion The Pf332-DBL region of Plasmodium falciparum membrane protein was successfully expressed in Pichia pastoris,which laid a foundation of developing subunit vaccine by using the protein.