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.
作者
伍苑宾
胡兢晶
王波
苏海浩
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)
出处
《热带医学杂志》
CAS
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)。
