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目的:观察密码子优化的SARS -CoVN基因的重组质粒,以及DNA初始免疫-蛋白加强免疫方式诱导小鼠的体液免疫应答水平,探讨提高SARS -CoVDNA免疫原性的途径。方法:SARS -CoVBJ01株N基因和密码子优化N基因分别通过从病毒RNA中RT -PCR扩增和人工合成获得,并克隆至pVAX1载体。将重组质粒DNA以 100μg/只的剂量肌肉注射免疫BALB/c小鼠,于第 20天同剂量再免疫 1次后,于第 40天再用 50μg/只剂量的N蛋白加强免疫。每次免疫后于第 10天采集血清,用ELISA检测血清中抗SARS- CoVN蛋白特异性抗体效价。同时设单纯重组质粒DNA免疫组和重组N蛋白免疫组作为对照。结果:密码子优化的N基因的重组质粒DNA免疫可诱导小鼠产生高水平体液免疫应答;而采用DNA初始免疫 -N蛋白加强免疫的方式则可使抗体效价提高至少 10倍。结论:密码子优化和DNA初始免疫-蛋白加强免疫这两种途径均可提高SARS CoVN基因重组质粒DNA在小鼠中的体液免疫应答水平。
OBJECTIVE: To observe the recombinant plasmid of codon-optimized SARS-CoVN gene and the level of humoral immune response induced by DNA initial immunization-protein boosting in mice, and to explore ways to improve the immunogenicity of SARS-CoV DNA. Methods: The N gene of SARS-CoVBJ01 strain and the codon-optimized N gene were obtained by RT-PCR amplification and artificial synthesis respectively from viral RNA and cloned into pVAX1 vector. BALB / c mice were immunized intramuscularly with the recombinant plasmid DNA at a dose of 100 μg / mouse, restimulated once with the same dose on the 20th day, and then boosted with a 50 μg / dose of the N protein on the 40th day. Serum was collected on the 10th day after each immunization and the serum anti-SARS-CoVN protein-specific antibody titers were measured by ELISA. At the same time, pure recombinant plasmid DNA immunization group and recombinant N protein immunization group as a control. RESULTS: Immunization with recombinant plasmid DNA of codon optimized N gene induced a high level of humoral immune response in mice. However, the antibody titer increased by at least 10-fold with DNA prime-N boost. Conclusion: Codon optimization and initial DNA immunization - protein boosting can increase the humoral immune response of recombinant plasmid DNA of SARS CoVN gene in mice.