小鼠Wnt-3a基因真核表达载体的构建及其在cos-7细胞中的表达

Construction of eukaryotic expression vector of mouse Wnt-3a gene and its expression in cos-7 cells

目的:克隆小鼠胚脑中Wnt-3a基因片段,构建pSecTag2／Hygro B-Wnt3a 真核表达载体,观察其在cos-7细胞中的表达,为基因治疗脊髓损伤提供实验依据。方法:实验于2004-09／2005-03在安徽医科大学分子生物学实验室完成。选取清洁级孕13．5 d的KM小鼠1只,脱颈处死,冰冷条件下迅速取出胚鼠,解剖显微镜下剥离胚脑,在无RNA酶污染的条件下迅速提取胚脑总RNA。利用反转录聚合酶链方法扩增小鼠胚脑Wnt-3a基因片段,将该基因片段克隆入T载体,聚合酶链反应法筛选并测序,双酶切后构建重组真核表达载体pSecTag2／Hygro B-Wnt3a。转染并筛选稳定表达的cos-7细胞,Western blot鉴定重组Wnt-3a蛋白的表达。结果:①反转录聚合酶链反应获得目的基因小鼠Wnt-3a cDNA:自胚鼠脑组织总RNA经反转录聚合酶链反应扩增后,凝胶电泳可见约1．1kb的特异性扩增片段,与预期获得产物大小相符。②pMD18-T/Wnt3a克隆质粒聚合酶链反应初步筛选与测序:测序报告所克隆的Wnt-3a为1 058 bp,通过Blast同源性分析,与GeneBank收录的序列一致,克隆小鼠Wnt-3a 基因获得成功。③真核表达载体pSecTag2／Hygro B-Wnt3a的双酶切及测序:随机挑选10个克隆,聚合酶链反应扩增筛选出阳性克隆5个,经 XhoⅠ和HindⅢ双酶切鉴定、测序及。Blast分析鉴定重组质粒构建成功。④Western Blot鉴定重组小鼠Wnt-3a蛋白在cos-7细胞中的表达:脂质体转染cos-7后48 h,Western Blot鉴定出在细胞内存在Wnt-3a蛋白的表达,转染pSecTag2／Hygro B-Wnt3a的cos-7细胞裂解液在45KD处出现阳性条带,而培养上清中未能检测到蛋白的表达。结论:小鼠胚脑Wnt-3a基因的真核表达载体pSecTag2／Hygro B-Wnt3a 构建成功,转染cos-7细胞后能够表达重组Wnt-3a蛋白。

AIM：To clone the Wnt-3a gene from mouse embryo brain to construct its eukaryotic expression vector pSecTag2/Hygro B-Wnt3a,and observe its expression in cos-7 cells so as to provide experimental evidence for gene therapy on spinal injury. METHODS：The experiment was completed in the molecular biologcial laboratory of Anhui Medical University between September 2004 and March 2005.A KM mouse of pregnancy for 13.5 days was selected and killed by dislocation of neck,the germinal mouse was quickly taken out under cold condition, and the embryo brain was stripped under anatomic microscope, and then the total RNA of embryo brain was quickly extracted under the condition of without RNA enzyme contamination.The complete encoded sequence of mouse Wnt-3a gene was amplified by reverse transcription-polymerase chain reaction（RT-PCR） from mouse embryo brain. The amplified fragment was clOned into T vector, and then inserted into eukaryotie expression vector pSeeTag2/Hygro B to construct the recombined plasmid that encoded Wnt-3a cDNA.Cos-7 cells were transfected mediated by liposome, and then the expression protein was detected by Western blot. RESULTS： ①The RT-PCR obtained mouse wnt-3a gene：After amplification by RT-PCR,agarose gel could observe the specific amplified fragment of 1.1 kb,which was concordant with the predicted size.②Then the target fragments were inserted into pMDI 8-T vector. After sequencing and Blast analysis, we successfully obtained the mouse wnt-3a gene. ③After selection by double cut with restriction endonuclease XhoⅠand Hind Ⅲ,eonfirmation by sequencing and Nucleotide homology analysis to GeneBank, the recombinant plasmid pSecTag2/Hygro B-Wnt3a for eukaryotic expression was also constructed. ④The reeombined eukaryotic expression vector pSecTag2/Hygro B-Wnt3a transfected cos-7 ceils by liposome reagent. 48 hours after transfection, the recombined Wnt-3a protein was detected,the specific 45 KD protein bands was only detected in cell lysates, not in supernatant. CONCLUSION：We successfully construct the eukaryotic expression vector of mouse Wnt-3a gene of mouse embryo brain. After transfecting cos-7 cells, the recombinant Wnt-3a protein is detected.