优化密码沙眼衣原体主要外膜蛋白基因的设计合成与表达

Design and expression of codon optimized chlamydia trachomatis major outer membrane protein gene

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 codon-optimized MoPn-maior outer membrane protein (MOMP) gene(HuMOMP),construct recombinant eukaryotic expression plasmids of HuMOMP gene and EGFP-HuMOMP fusion gene. Methods: The differences of preferable codon usage between chlamydia muridarum(MoPn) and mammal gene were analyzed,and the sequence of codon-optimized MoPn-MOMP (HuMOMP) was designed and synthesized. Plasmid pUC-HuMOMP was digested with KpnI and EcoRI to get HuMOMP segment,which was cloned into vector pcDNA3 to construct pcDNA3-HuMOMP. Plasmid pcDNA3-HuMOMP was digested with KpnI and ApaI to get HuMOMP segment,which was cloned into vector pEGFP-C1 to construct pEGFP-HuMOMP. After confirming its correctness by restriction enzyme analysis,pEGFP-HuMOMP was transfected into COS-1 cell instantly with liposome,and observed its expression using fluorescence microscope after 24 hours. Results: Codon-optimized MoPn-MOMP gene (HuMOMP) was designed and synthesized,then verified by gene sequencing. The recombinant plasmids pcDNA3- HuMOMP and pEGFP-HuMOMP digested with corresponding restriction enzymes could get correct DNA segments. EGFP -HuMOMP fusion gene was expressed effectively in COS-1 cells. Fluorescent protein mainly existed in cytoplasm,which further certified the sequence precision of HuMOMP gene and the constructive correction of fluorescent expression plasmids. Conclusion: Designed and synthesized codon-optimized MoPn-MOMP gene (HuMOMP) can be expressed in mammalian cell successfully.