摘要
目的构建EBA-175Ⅱ区F2结构域基因的原核表达载体。方法采用PCR的方法从恶性疟原虫基因组DNA中扩增出EBA-175Ⅱ区F2结构域856～1848bp基因,定向克隆入PMD18-T质粒,再经BamHⅠ和XhoⅠ酶切亚克隆入高效表达载体pET23a(+)。重组质粒经PCR、酶切、测序鉴定后,在BL21(DE3)菌株中进行IPTG诱导表达。结果重组质粒经序列测定证实插入基因为目的基因,符合表达框架,经IPTG诱导,在BL21(DE3)菌株中以非融合蛋白形式表达,SDS-PAGE电泳显示在约36.4kDa处可见明显蛋白增粗条带,与预期结果一致。结论成功构建了重组EBA-175Ⅱ区F2结构域基因表达载体,并在BL21(DE3)中有效表达。
Objective To construct recombinant expressio n vector containing the F2domain located within regionⅡof the 175kDa Plasmodium falciparum erythrocyte binding antigen(EBA-175).Methods The 856-1848bp fragment of EBA-175was amplified with PCR.The P CR product was directionally cloned into PMD18-T.The insert was excised by BamHⅠand XhoⅠand subcloned into high expression v ector pET23a +.The recombinant protein is induced to express in BL21(DE3)transformed cells by IPTG.Results The inserted fragment in pET23a +was confirmed to be the target gene.With induction of IPTG,a new protein with the expected molecular mass of 36.4kDa was expressed.Conclusion The recombinant expression vector was c onstructed successfully,which cou ld express the recombinant EBA-175p rotein in BL21(DE3)cells.
出处
《热带医学杂志》
CAS
2005年第2期128-130,115,共4页
Journal of Tropical Medicine
基金
国家自然科学基金(No.30170839)。
