WISP3基因突变体的构建及在COS-7细胞中的表达

目的:通过构建晚发型脊柱骨骺发育不良伴进行性骨关节病(spondyloepiphyseal dysplasia tarda with progressive arthropathy,SEDT-PA)致病型(1000T/C,840 delT)Wnt诱导分泌蛋白3(wnt-inducible secreted protein 3,WISP3)的真核表达载体,并将其表达于真核表达系统COS-7细胞系,为研究WISP3突变致SEDT-PA的机制奠定基础.方法:从正常人软骨细胞获得野生型WISP3基因的全长cDNA(WT-WISP3):用定点突变方法构建携带WISP3基因致病型的重组真核表达质粒MUT1000T/C/pcDNA3.1( )和MUT840delT/pcDNA3.1( );以空白载体pcDNA3.1( )为对照,脂质体转染法将重组质粒瞬时转染COS-7细胞,48 h后提取转染细胞的总RNA和总蛋白;半定量RT-PCR和免疫印迹方法检测WISP3基因的表达.结果:经测序证实,构建的野生型和突变型WISP3基因的全长cDNA序列分别与文献及前期报道的SEDT-PA患者WISP3基因突变类型一致;酶切鉴定证实,成功构建了3个重组真核表达质粒WT-WISP3/pcDNA3.1( ),MUT1000T/C/pcDNA3.1( )及MUT840delT/pcDNA3.1( )];半定量RT-PCR和免疫印迹示,重组质粒在COS-7细胞中均高效表达.结论:成功构建了SEDT-PA致病基因WISP3的突变体并在COS-7细胞中得到表达,为进一步探讨SEDT-PA的发病机制创造了条件.

Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells

Objective To construct two types of Wnt-inducible secreted protein 3(WISP3)gene's mutants(1000T/C,840delT)found in spondyloepiphyseal dysplasia tarda with progressive anthopathy(SEDT-PA)patients,and to observe their expression in COS-7 cells.Methods Full-length cDNA of wild type WISP3 gene(WT-WISP3)was amplified from human chondrocytes by RT-PCR,and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT 1000T/C and MUT 840delT).The recombined plasmids WT-WISP3/pcDNA3.1( ),MUT 1000T/C /pcDNA3.1( )and MUT 840delT /pcDNA3.1( )were transfected transiently into COS-7 cells by liposome-mediated method,and pcDNA3.1( )vector was used as a control.The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection.The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot.Results By restriction endonuclease analysis and sequencing,the sequence of MUT 1000T/C and MUT 840delT were consistent with that mutated in SEDT-PA,and the open reading frames matched with the vector sequence.Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells.Conclusion WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination,and expressed in COS-7 cells,which lays the foundation for the further study on its molecular functions in SEDT-PA.