WISP3基因突变体的构建及在COS-7细胞中的表达  

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

作　　者：王敏[1] 彭依群[1] 周后德[1] 翟木绪[1] 何玉玲[1] 谢辉[1] 罗湘杭[1] 郭丽娟[1] 廖二元[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所,长沙410011

出　　处：《中南大学学报（医学版）》2008年第1期8-15,共8页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30400218)~~

摘　　要：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 dysplasiatarda with progressive arthropathy,SEDT-PA)致病型(1000T/C,840delT)Wnt诱导分泌蛋白3(Wnt-induciblesecreted protein3,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,48h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。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 Fulllength eDNA 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 ceils by liposome-mediated method, and pcDNA3. 1 （ ＋ ） vector was used as a control. The total RNA and protein of the transfected COS-7 ceils were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 ceils 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 ceils. Conclusion WISP3 gene＇ s mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 ceils, which lays the foundation for the further study on its molecular functions in SEDT-PA.

关 键 词：晚发型脊柱骨骺发育不良伴进行性骨关节病 Wnt诱导分泌蛋白3 突变体 定点突变 

分 类 号：R681.1[医药卫生—骨科学]