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

作　　者：黄立敏 张春丽[1] 李泽雨 孙晋 田红卫 HUANG Limin;ZHANG Chunli;LI Zeyu;SUN Jin;TIAN Hongwei(National and Local Joint Engineering Center for Biodiag-nosis and Treatment,Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China;Department of Geriatric Hepatobi-liary,Pancreatic and Spleen Surgery,Second Affiliated Hospital of Xi'an Jiaotong University)

机构地区：[1]西安交通大学第二附属医院生物诊断治疗国家地方联合工程中心,西安710004 [2]西安交通大学第二附属医院肝胆胰脾外科

出　　处：《山西医科大学学报》2025年第1期20-27,共8页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81802456);陕西省自然科学基金面上项目(2024JC-YBMS-691)。

摘　　要：目的 分别构建人源RNA解旋酶DHX34基因不同功能结构域缺失的突变载体,测序分析和验证其在293T细胞中的蛋白表达。方法 以pECMV-hDHX34-3×FLAG-SV40-Puro(hDHX34)质粒为模板,根据同源重组克隆的技术原理设计不同功能结构域引物,通过聚合酶链式反应扩增片段,重组克隆到pECMV-MCS-3×FLAG-SV40-Puro空载上,经限制性酶切、重组、转化分别构建ΔNTD、ΔCTD、ΔRecA1、ΔRecA2、ΔHelicase、ΔOB、ΔWR载体。经测序验证后,利用脂质体转染293T细胞,Western blot检测各载体蛋白的表达。结果 测序结果与预期结果相符,证实人源RNA解旋酶DHX34基因不同功能结构域缺失的真核表达载体构建成功。核酸电泳结果显示,hDHX34及突变质粒ΔNTD、ΔCTD、ΔRecA1、ΔRecA2、ΔHelicase、ΔWR和ΔOB片段长度符合预期,证明质粒构建成功。Western blot结果显示,293T细胞分别转染ΔNTD、ΔCTD、ΔRecA1、ΔRecA2、ΔHelicase、ΔWR、ΔOB载体后,蛋白条带清晰可见且符合预期相对分子量,说明质粒转染成功,hDHX34及各突变质粒成功表达。结论成功构建人源RNA解旋酶DHX34基因不同功能结构域缺失的突变载体,并验证了各载体的表达情况,为研究人源DHX34基因的生物学功能奠定基础。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,includingΔNTD,ΔCTD,ΔRecA1,ΔRecA2,ΔHelicase,andΔOB,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,andΔOB)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 theΔNTD,ΔCTD,ΔRecA1,ΔRecA2,ΔHelicase,ΔWR,andΔOB 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 

分 类 号：Q78[生物学—分子生物学]