RNA解旋酶DHX37基因不同功能结构域载体的构建及鉴定OA

Objective:To discuss the functional domains of the human RNA helicase DHX37 gene predicted by online websites and software,to construct its different functional domain deletion vectors,and to verify the correctness of the constructed vectors 27.Methods:Using the human DEAH-box helicase 37(hDHX37)plasmid as a template,based on homologous recombination cloning technology,SnapGene V 6.0.2 software was used to design functional domain-specific primers for the truncations ATP binding domain(ATP binding),C-terminal domain(CTD),HA2 subunit(HA2),and Oligos/Accharide binding fold domain(O/A binding fold);prime STAR GXL polymerase,a high-fidelity DNA polymerase,was used to perform PCR amplification of the truncated target fragments ATP binding,c-terminal-1,c-terminal-2,HA2-1,HA2-2,and O/A binding;a recombination cloning kit was used to directionally clone the truncated fragments into the double-digested pCMV-MCS-3×HA-Neo empty vector;after transforming the recombinant vectors into E.coli DH5α competent cells,the correctness of the functional domain vectors was verified by restriction enzyme Hind Ⅲ digestion,agarose nucleic acid gel electrophoresis,and DNA sequencing.Results:The lengths of the target fragments amplified by PCR,ATP binding(2 148 bp),c-terminal-1(1 326 bp),c-terminal-2(1 269 bp),HA2-1(2 205 bp),HA2-2(894 bp),and O/A binding fold(2 583 bp),were consistent with the design;Hind Ⅲ single enzyme digestion of the mutant plasmids ΔATP binding,Δc-terminal,ΔHA2,and ΔO/A binding fold yielded linearized DNA fragments with lengths of 8 939,7 589,8 036,8 540,and 8 027 bp,respectively,and the product band sizes all matched the design.The DNA sequencing Blast comparative analysis results showed that ΔATP binding lacked the ATP binding domain(1-442),Δc-terminal lacked the c-terminal domain(443-735),ΔHA2 lacked the HA2 domain(736-861),and ΔO/A binding fold lacked the O/A binding fold domain(862-1 158),with deletions of 1 326,879,375,and 891 bp,respectively;the positions and lengths of the deleted domains in each mutant plasmid were completely consistent with the design.Conclusion:The four different functional domain vectors of human DHX37 gene,Δ ATP binding,Δc-terminal,ΔHA2,and ΔO/A binding fold,are successfully constructed.