密码修饰可增强沙眼衣原体MOMP DNA质粒免疫小鼠的免疫应答

Codon modification of the Chlamydia trachomatis MOMP gene enhances the immune responses in DNA-vaccinated mice

目的探讨密码修饰对沙眼衣原体主要外膜蛋白(MOMP)基因表达和DNA质粒免疫的影响。方法根据人类密码子使用偏好对鼠肺炎沙眼衣原体(Chlamydia trachomatis mouse pneumoni-tis)MOMP基因进行优化修饰,设计合成人源化MOMP基因(HuMOMP)。构建以pcDNA3为载体的含HuMOMP及含野生型MOMP基因(WtMOMP)的真核表达质粒。瞬时转染COS-1细胞,Western blot方法比较HuMOMP基因及WtMOMP基因在哺乳动物细胞中的蛋白表达水平。DNA质粒肌内免疫BALB/c小鼠,检测小鼠血清特异性抗体、DTH和淋巴细胞增殖反应,比较优化密码MOMP基因和野生型MOMP基因DNA质粒免疫的免疫效果。结果人工合成HuMOMP基因,成功构建重组真核表达质粒pcDNA3-HuMOMP及pcDNA3-WtMOMP。转染细胞Western blot结果显示,HuMOMP基因的蛋白表达水平明显高于WtMOMP基因。ELISA结果表明,HuMOMP DNA质粒免疫组小鼠血清特异性IgG抗体有所升高;细胞免疫检测结果显示,HuMOMP DNA质粒免疫组小鼠足垫肿胀程度增强、淋巴细胞增殖实验刺激指数(SI)增高,两者与WtMOMP DNA质粒免疫组相比P<0.05,差异均有统计学意义。结论人源化密码修饰能够增强沙眼衣原体MOMP基因在哺乳动物细胞中的蛋白表达及DNA质粒免疫小鼠的免疫反应,这对于新型衣原体DNA疫苗的研究具有重要意义。

Objeclive To analyze the effects of eodon modification on the expression and immunogenieity of DNA candidate vaccines encoding the Chlamydia trachomatis mouse pneumouitis （MoPn）, major outer membrane protein（MOMP）. Methods The DNA sequence encoding mature MOMP of C. trachomatis MoPn was moditied to substitute human preferred eodon for the rarely used eedon. The humanized MOMP gene （HuMOMP） was eloued into a mammalian expression vector pcDNA3. The in vitro expression of HuMOMP and WtMOMP gene in COS- 1 cells were compared by Western blot. The immunogenicity of the two constructs was examined by measuring the immune responses in the intramuscularly immunized BALB/c mice including MoPn-EB specific antibody production, DTH reaction and lymphocyte proliferation, Results The HuMOMP gene was designed and synthesized based on the differences in preferable codon usage between MoPn and human genomes. The correctness of the synthesized HuMOMP was verified by gene sequencing. In vitro experiments showed COS- 1 cells transfected with HuMOMP gene produced significantly higher levels of MOMP protein than those transfected with WtMOMP. In vivo study showed that mice immunized with HuMOMP produced higher MoPn-specific IgG antibody than those with WtMOMP immunization. DTH reaction was significantly higher in HuMOMP immunized group than that in WtMOMP immunized group（ P 〈0.05）. Furthermore, lymphocytes from HuMOMP immunized group showed stronger proliferative responses than those from WtMOMP immunized group following MoPn-specific stimulation （ P 〈 0.05 ）. Conclusion Humanized codon optimization can significantly enhance the gene expression. Both humoral and cellular immunogenicity of MoPn MOMP DNA vaccine are improved.