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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。
OBJECTIVE: To clone the Wnt-3a gene fragment in mouse embryonic brain and construct the eukaryotic expression vector pSecTag2 / Hygro B-Wnt3a. The expression of cosmid gene in cos-7 cells was observed to provide experimental evidence for gene therapy of spinal cord injury. Methods: The experiment was performed in molecular biology laboratory of Anhui Medical University from September 2004 to March 2005. One KM mouse of clean grade pregnant 13.5d was selected and killed by atrophy. The embryos were removed rapidly under the condition of cold and dissected from the embryonic brain under a dissecting microscope. The total RNA of embryo brain was extracted rapidly without RNase contamination. The Wnt-3a gene fragment of mouse embryonic brain was amplified by reverse transcription-polymerase chain reaction. The gene fragment was cloned into T vector and screened by polymerase chain reaction and sequenced. After double enzyme digestion, the recombinant eukaryotic expression vector pSecTag2 / Hygro B-Wnt3a. The stable expression of cos-7 cells was transfected and screened, and the expression of recombinant Wnt-3a protein was identified by Western blot. Results: (1) Wnt-3a cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The total RNA was extracted from the embryonic rat brain by reverse transcription polymerase chain reaction The fragment was amplified, consistent with the expected size of the product obtained. ② Preliminary screening and sequencing of the pMD18-T / Wnt3a cloning plasmid PCR: The cloned Wnt-3a gene was 1 058 bp, which was identical to the sequence of GeneBank by Blast homology analysis. The Wnt-3a gene was cloned Success. (3) Double enzyme digestion and sequencing of eukaryotic expression vector pSecTag2 / Hygro B-Wnt3a: Ten clones were randomly selected and 5 positive clones were screened by polymerase chain reaction (PCR). Blast analysis identified that the recombinant plasmid was successfully constructed. Western Blot to identify the recombinant mouse Wnt-3a protein expression in cos-7 cells: liposome transfection COS-7 48h, Western Blot identified the presence of Wnt-3a protein expression in cells transfected with pSecTag2 / The positive band of Hygro B-Wnt3a cos-7 cell lysate appeared at 45KD, but the protein expression was not detected in the culture supernatant. CONCLUSION: The eukaryotic expression vector pSecTag2 / Hygro B-Wnt3a of mouse embryonic brain Wnt-3a gene was constructed successfully and the recombinant Wnt-3a protein can be expressed after transfection into cos-7 cells.