山西医科大学学报2025，Vol.56Issue(1)：20-27,8.DOI:10.13753/j.issn.1007-6611.2025.01.004
人源RNA解旋酶DHX34基因功能结构域缺失载体的构建及鉴定
Construction and identification of a functional domain defective vector of human RNA helicase DHX34 gene
摘要
Abstract
Objective To construct mutant vectors with deletion of different functional domains of human RNA helicase DHX34 gene,and confirm the protein expression in 293T cells by sequencing.Methods Using the plasmid pECMV-hDHX34-3×FLAG-SV40-Puro as a template,the primers targeting various protein domains were designed based on the principles of homologous recombi-nation cloning.The corresponding fragments were amplified by polymerase chain reaction(PCR)and cloned into the vector of pECMV-MCS-3×FLAG-SV40-Puro.Mutant vectors,including ΔNTD,ΔCTD,ΔRecA1,ΔRecA2,ΔHelicase,and ΔOB,were constructed after restriction enzymes,recombination,and transformation.After verification by sequencing,293T cells were transfected using lipo-somes,and the protein expression of each mutant was assessed by Western blot.Results The results of sequencing were consistent with the expected outcomes,which confirmed the successful construction of eukaryotic expression vector with deletions in various func-tional domains of human RNA helicase DHX34 gene.The results of nucleic acid electrophoresis revealed that the sizes of hDHX34 and mutant plasmids(ΔNTD,ΔCTD,ΔRecA1,ΔRecA2,ΔHelicase,ΔWR,and ΔOB)matched the expected values,which further con-firmed the successful construction of the plasmids.Western blot results showed that the clearly visible protein bands were observed at the expected molecular weights in 293T cells transfected with the ΔNTD,ΔCTD,ΔRecA1,ΔRecA2,ΔHelicase,ΔWR,and ΔOB vectors,indicating successful expression of both hDHX34 and mutant proteins.Conclusion Mutant vectors with deletion of different functional domains of the human RNA helicase DHX34 gene are successfully constructed,and the expression of each vector is con-firmed.These results provide a solid foundation for further investigation into the biological functions of the hDHX34 gene.关键词
RNA解旋酶/DHX34基因/功能结构域缺失/载体构建/重组克隆/限制性内切酶Key words
RNA helicase/DHX34 gene/deletion of functional domains/vector construction/recombinant clones/restriction enzymes分类
生物科学引用本文复制引用
黄立敏,张春丽,李泽雨,孙晋,田红卫..人源RNA解旋酶DHX34基因功能结构域缺失载体的构建及鉴定[J].山西医科大学学报,2025,56(1):20-27,8.基金项目
国家自然科学基金资助项目(81802456) （81802456）
陕西省自然科学基金面上项目(2024JC-YBMS-691) （2024JC-YBMS-691）
