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

作　　者：孔祥礼 燕贺[1] 涂宏[1] 冯欣宇[1] 丰俊[1,3] 夏志贵[1] 周水森 KONG Xiang-li;YAN He;TU Hong;FENG Xin-yu;FENG Jun;XIA Zhi-gui;ZHOU Shui-sen(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,NHC Key Laboratory of Parasite and Vector Biology,National Center for International Research on Tropical Diseases,Ministry of Science and Technology,WHO Collaborating Centre for Tropical Diseases,Shanghai 200025,China;Shandong Institute of Parasitic Diseases,Shandong First Medical University&Shandong Academy of Medical Sciences;Chinese Center for Tropical Diseases Research,School of Medicine,Shanghai Jiao Tong University)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,科技部热带病国际合作中心,世界卫生组织热带病合作中心,上海200025 [2]山东省寄生虫病防治研究所,山东第一医科大学(山东省医学科学院) [3]上海交通大学医学院-国家热带病研究中心

出　　处：《中国病原生物学杂志》2021年第4期442-445,460,共5页Journal of Pathogen Biology

基　　金：上海市卫生健康委员会临床专项面上课题(No.202040049);山东省自然科学基金项目(No.ZR2019PH118)。

摘　　要：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.

关 键 词：疟原虫 恶性 Kelch 13螺旋桨蛋白 F446I位点 杆状病毒 真核表达 

分 类 号：R382.31[医药卫生—医学寄生虫学]