人细小病毒B19基因组编码蛋白真核表达质粒的构建与鉴定  

Construction and Identification of Eukaryotic Expression Plasmids of Human parvovirus B19 Genome-Encoded Proteins

作　　者：钟亚迪 贾俊婷[1] 范瑞 马丽敏 马玉媛[1] 章金刚[1] ZHONG Ya-Di;JIA Jun-Ting;FAN Rui;MA Li-Min;MA Yu-Yuan;ZHANG Jin-Gang(Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China)

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2017年第5期618-622,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81570169);北京市自然科学基金(7162147)

摘　　要：目的:构建人细小病毒B19(B19V)基因组编码蛋白的真核表达质粒,并对其表达进行鉴定,为研究B19V蛋白功能提供支持。方法:设计并合成B19V基因组编码蛋白VP1、VP2、NS1、11k Da、X、7.5k Da和VP1独特区域(VP1u)的PCR扩增引物,以B19V感染性克隆p B19-M20为模板,PCR扩增基因组编码蛋白的核苷酸序列,将其插入真核表达载体pcDNA3.1(+)-HA-Flag,并进行酶切鉴定和核苷酸序列测定;将鉴定正确的重组质粒转染HEK293T细胞,通过免疫印迹分别检测病毒蛋白的表达情况。结果:构建了B19V 7种蛋白的真核表达质粒,免疫印迹鉴定表明均可表达相应的目的蛋白。结论:构建了B19V 7种蛋白的真核表达质粒,为深入研究B19V蛋白功能及其在致病机制中的作用奠定了基础。Objective:To construct and identify eukaryotic expression plasmid of Human parvovirus B19(B19V)genome-encoded proteins to support the functional research of the proteins.Methods:First of all,the coding sequences of B19V proteins were amplified from B19V infectious clone pB19-M20by PCR method using specificprimers.Secondly,the amplicons were cloned into the pcDNA3.1(+)-HA-Flag vector respectively,and then thepositive clones were confirmed by restriction digestion and sequencing.Finally,the constructed recombinant plasmids containing the target genes were transfected into HEK293T cells,followed by Western blot analysis for targetproteins expression.Results:Recombinant eukaryotic expression plasmids of B19V genome-encoded proteins wereconstructed and their expression was confirmed in HEK293T cells.Conclusion:Our study has laid a foundationfor further exploring the function of B19V proteins and their roles in the pathogenesis of B19V associated diseases.

关 键 词：人细小病毒B19 病毒蛋白 真核表达质粒 

分 类 号：Q78[生物学—分子生物学]