恶性疟原虫pfK13-F446I基因重组杆状病毒质粒的构建及其在昆虫细胞SF9中的表达

Construction and expression of a recombinant Baculovirus plasmid with the K13-F446I gene of Plasmodium falciparumin Spodoptera frugiperda 9cells

目的构建恶性疟原虫Kelch 13-F446I(pfK13-F446I)基因重组杆状病毒质粒,并在昆虫细胞SF9中表达。方法体外合成恶性疟原虫Kelch 13野生型和F446I蛋白结构域片段碱基,定向克隆至转移载体pFastBac,构建pfK13-WT-Bac和pfK13-F446I-Bac质粒,转化大肠埃希菌DH10Bac感受态细胞后进行基因重组。提取重组病毒,PCR和Western blot验证后转染草地贪夜蛾细胞(SF9)。收集亲代重组病毒反复感染SF9细胞进行病毒扩增,优化可溶性蛋白的纯化条件,采用SDS-PAGE电泳分析重组蛋白表达情况。结果构建了含恶性疟原虫pfK13-F446I基因的重组质粒pfK13-F446I-Bac,大小约3 430 bp。转染SF9细胞后获得有感染力的重组杆状病毒,并在SF9细胞中表达pfK13-F446I蛋白,经梯度洗脱、去标签、透析等获得大小为44 ku的单一电泳条带的pfK13-F446I蛋白。结论成功构建pfK13-F446I基因重组杆状病毒质粒,并在昆虫细胞SF9中进行成功表达。

Objective To express the soluble recombinant Plasmodium falciparum K13-F446 I(pfK13-F446 I) gene in Spodoptera frugiperda 9(SF9) cells. Methods Kelch 13 of wild-type P. falciparum and fragments from F446 I mutants were synthesized in vitro and cloned into the transfer vector pFastBac to construct the plasmids pfK13-WT-Bac and pfK13-F446 I-Bac. The plasmids were transformed into human Escherichia coli DH10 Bac competent cells after genetic recombination. The recombinant virus was extracted and verified as correct using PCR and Western blotting. The following recombinant virus was then transfected into SF9 cells. The parental recombinant virus was collected and repeatedly used to infect SF9 cells for virus amplification to examine the conditions for purification of the soluble protein. Expression of the recombinant protein was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Results A recombinant pfK13-F446 I-Bac plasmid containing the P. falciparum pfK13-F446 I gene was constructed. It was about 3,430 bp in length. After its transfection into SF9 cells, an infectious recombinant baculovirus was obtained, and the pfK13-F446 I protein was successfully expressed in SF9 cells. Pure pfK13-F446 I protein with a molecular mass of 44 ku was obtained through gradient elution, detagging, and dialysis. Conclusion A baculovirus plasmid for expression of a recombinant pfK13-F446 I gene was successfully constructed, and the gene was expressed in SF9 cells.