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目的:设计合成优化密码沙眼衣原体主要外膜蛋白(MOMP)基因,克隆构建优化密码MOMP(HuMOMP)基因的真核表达质粒及EGFP-HuMOMP融合基因表达质粒。方法:利用软件分析比较鼠肺炎沙眼衣原体(MoPn)与人类等哺乳动物基因组密码子使用的差异,根据所得数据对MOMP基因进行优化设计与合成。双酶切pUC-HuMOMP质粒,游离HuMOMP片段,定向克隆入pcDNA3的相应位点,构建重组真核表达质粒pcDNA3-HuMOMP;双酶切游离pcDNA3-HuMOMP中的HuMOMP片段,定向克隆入pEGFP-C1的相应位点,构建重组真核表达质粒pEGFP-HuMOMP;pEGFP-HuMOMP质粒体外脂质体转染COS-1细胞,24h后观察荧光蛋白的表达。结果:合成的HuMOMP基因全长1125bp,经DNA测序无误。酶切鉴定pcDNA3-HuMOMP及pEGFP-HuMOMP质粒获得预期分子量DNA片段。EGFP-HuMOMP融合基因在COS-1细胞中获得明显表达,表达产物定位于胞浆。结论:设计合成的优化密码HuMOMP基因能够在哺乳动物细胞中成功表达。
OBJECTIVE: To design and synthesize the major outer membrane protein (MOMP) gene of Chlamydia trachomatis and to construct the eukaryotic expression plasmid of optimized MOMP (HuMOMP) gene and EGFP-HuMOMP fusion gene expression plasmid. METHODS: Software was used to analyze the differences in codon usage between mammals such as C. pneumoniae and humans, and to optimize MOMP gene design and synthesis based on the data obtained. The pUC-HuMOMP plasmid and the free HuMOMP fragment were double-digested and cloned into the corresponding sites of pcDNA3 to construct the recombinant eukaryotic expression plasmid pcDNA3-HuMOMP. The HuMOMP fragment was digested by double digestion of pcDNA3-HuMOMP and cloned into pEGFP-C1 The recombinant plasmid pEGFP-HuMOMP was constructed. The pEGFP-HuMOMP plasmid was transfected into COS-1 cells in vitro, and the expression of fluorescent protein was observed 24h later. Results: The synthesized HuMOMP gene was 1125bp in length and was sequenced by DNA. The pcDNA3-HuMOMP and pEGFP-HuMOMP plasmids were identified by restriction enzyme digestion to obtain DNA fragments of expected molecular weight. The EGFP-HuMOMP fusion gene was expressed in COS-1 cells and the expression product localized in the cytoplasm. Conclusion: The designed optimized HuMOMP gene can be successfully expressed in mammalian cells.