靶向人巨细胞病毒UL145基因的CRISPR/Cas9基因敲除质粒的构建  

Construction of CRISPR/Cas9 gene knockout plasmid targeting human cytomegalovirus UL145 gene

作　　者：伍苑宾[1] 胡兢晶[1] 王波[1] 苏海浩[1] 谭琪琪 WU Yuan-bin;HU Jing-jing;WANG Bo;SU Hai-hao;TAN Qi-qi(Department of Pediatric,Guangdong Women and Children Hospital,Guangzhou,Guangdong 511442,China)

机构地区：[1]广东省妇幼保健院儿科,广东广州511442

出　　处：《热带医学杂志》2021年第1期10-12,17,F0003,共5页Journal of Tropical Medicine

基　　金：广东省自然科学基金(2016A030313788)。

摘　　要：目的构建靶向人巨细胞病毒UL145基因的CRISPR/Cas9基因敲除质粒。方法根据CRISPR/Cas9设计原则,设计3条靶向UL145基因的gRNA,构建重组lenti CRISPR-UL145-T1、T2、T3表达质粒,转化并挑选阳性克隆,进行PCR及测序验证。同时通过荧光素酶SSA(Luciferase SSA)检测CRISPR/Cas9活性,筛选出载体活性最佳质粒。结果设计了3条靶向gRNA并构建lenti CRISPR-UL145-T1、T2、T3表达质粒,经PCR及测序鉴定载体构建成功;通过Luciferase SSA检测,结果显示,lenti CRISPR-UL145-T3载体活性最强,明显高于lenti CRISPR-UL145-T1、T2载体活性。结论重组质粒lenti CRISPR-UL145-T3筛选为活性最佳质粒,为进一步进行体内外敲除人巨细胞病毒UL145基因及其功能奠定基础。Objective To construct a CRISPR/Cas9 gene knockout plasmid targeting human cytomegalovirus UL145 gene.Methods According to CRISPR/Cas9 design principles,three g RNAs targeting the UL145 gene were designed;constructed recombinant lenti CRISPR-UL145-T1,T2,T3 expression plasmids were used to transform cells;positive cloneswere selected from the transformed cells for PCR and sequencing verification.CRISPR/cas9 activity was detected byLuciferase SSA,and the plasmid with the best vector activity was selected.Results Three targeted g RNAs were designedand used for the construction of lenti CRISPR-UL145-T1,T2,T3 expression plasmids,which were verified by PCR andsequencing;the recombinant plasmid lenti CRISPR-UL145-T3 was screened as the most active plasmid by Luciferase SSAdetection.Conclusion The recombinant plasmid lenti CRISPR-UL145-T3 is screened as the most active plasmid,layingthe foundation for further knockout of human cytomegalovirus UL145 gene and its function in vivo and in vitro.

关 键 词：人巨细胞病毒 UL145 CRISPR/Cas9 基因敲除 

分 类 号：R373.9[医药卫生—病原生物学]