Zheng Yan, Zhao Wei-Ming, Wang Hong, Zhou Ya-Bin, Luan Yi, Qi Mei, Cheng Yi-Zhe, Tang Wei, Liu Juan, Yu Han, Yu Xiu-Ping, Fan Yi-Jun, Yang Xi
Department of Medical Microbiology, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China.
Biochem Cell Biol. 2007 Apr;85(2):218-26. doi: 10.1139/o06-211.
Chlamydia trachomatis is a kind of obligate intracellular bacterial pathogen that causes ocular and sexually transmitted diseases. In this study, we analyzed the codon usage patterns of the C. trachomatis mouse pneumonitis biovar (MoPn) and Homo sapiens. We found large differences between MoPn and human codon usages. To enhance the expression of Chlamydia protein in mammalian cells, the DNA sequence encoding the major outer-membrane protein (MOMP) of MoPn was modified to substitute the human-preferred codons for rarely used codons. The huma-optimized MOMP gene was synthesized and cloned into the pcDNA3 vector, as was the wild-type MOMP gene. The protein expression levels of the human-optimized MOMP and wild-type MOMP genes were compared. The experiments showed that the human-optimized MOMP gene produced significantly higher levels of MOMP protein than the wild-type MOMP, both in vitro and in vivo, but no obvious difference was observed in the levels of modified and native MOMP mRNA expression. The immunogenicity of the 2 constructs was examined using BALB/c mice following intramuscular immunization. The results showed that the mice immunized with the human-optimized MOMP produced higher levels of antigen-specific IgG antibody and showed stronger delayed-type hypersensitivity reactions and proliferative T cell responses than those immunized with the wild-type MOMP. Antigen-specific stimulation of spleen cells obtained from human MOMP DNA immunized mice produced higher levels of interferon-gamma than those obtained from wild-type MOMP DNA immunized mice. Taken together, the data show that human-optimized codon optimization can significantly enhance the gene expression and immunogenicity of the C. trachomatis MOMP DNA vaccine.
沙眼衣原体是一种专性胞内细菌病原体，可导致眼部疾病和性传播疾病。在本研究中，我们分析了沙眼衣原体小鼠肺炎生物变种（MoPn）和人类的密码子使用模式。我们发现MoPn和人类的密码子使用存在很大差异。为了增强衣原体蛋白在哺乳动物细胞中的表达，编码MoPn主要外膜蛋白（MOMP）的DNA序列被修饰，用人类偏好的密码子替换很少使用的密码子。合成了人类优化的MOMP基因并将其克隆到pcDNA3载体中，野生型MOMP基因也进行了同样操作。比较了人类优化的MOMP和野生型MOMP基因的蛋白质表达水平。实验表明，无论是在体外还是体内，人类优化的MOMP基因产生的MOMP蛋白水平均显著高于野生型MOMP，但修饰后的MOMP和天然MOMP mRNA表达水平未观察到明显差异。在肌肉注射免疫后，使用BALB/c小鼠检测了这两种构建体的免疫原性。结果显示，与野生型MOMP免疫的小鼠相比，用人类优化的MOMP免疫的小鼠产生了更高水平的抗原特异性IgG抗体，表现出更强的迟发型超敏反应和增殖性T细胞反应。从人类MOMP DNA免疫小鼠获得的脾细胞经抗原特异性刺激后产生的干扰素-γ水平高于野生型MOMP DNA免疫小鼠。综上所述，数据表明人类优化的密码子优化可显著增强沙眼衣原体MOMP DNA疫苗的基因表达和免疫原性。
