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目的:构建能表达密码子优化的中国HIV-1流行亚型AE、BC和B’gp120共识序列的核酸疫苗,并研究其免疫原性。方法:根据HIV-1的分子流行病学资料和包膜糖蛋白的氨基酸序列分析,设计了针对中国流行的AE,BC和B’(ThB)亚型的gp120共识序列。按哺乳动物细胞偏好的密码子对AE,BC和ThB3个亚型的gp120共识序列分别进行密码子优化,合成优化基因。将密码子优化的gp120基因克隆到核酸疫苗载体pSW3891,构建重组质粒核酸疫苗。将重组质粒转染293T细胞,并以重组质粒疫苗免疫新西兰兔。应用蛋白质免疫印迹法检测转染细胞中gp120蛋白的表达。ELISA方法检测免疫后兔血清中gp120特异性抗体的产生。结果:3个密码子优化的gp120核酸疫苗,均能正确表达gp120。这些gp120核酸疫苗免疫家兔后,能产生HIV-1包膜糖蛋白特异性的抗体。结论:成功构建3个密码子优化的、表达HIV-1中国流行亚型AE,BC和ThB的共识序列的gp120核酸疫苗,能诱导HIV-1包膜蛋白特异的免疫反应。
OBJECTIVE: To construct a nucleic acid vaccine capable of expressing codon-optimized consensus sequences of HIV-1 subtypes AE, BC and B’gp120 in China and investigate its immunogenicity. Methods: According to the molecular epidemiological data of HIV-1 and amino acid sequence analysis of envelope glycoprotein, consensus sequences of gp120 targeting AE, BC and B ’(ThB) subtypes of China were designed. Consensus sequences of gp120 of AE, BC and ThB3 isoforms were codon-optimized by mammalian cell-preferred codons, respectively, to synthesize optimized genes. The codon-optimized gp120 gene was cloned into the nucleic acid vaccine vector pSW3891 to construct a recombinant plasmid DNA vaccine. The recombinant plasmids were transfected into 293T cells and the New Zealand rabbits were immunized with recombinant plasmids. The expression of gp120 protein in transfected cells was detected by Western blotting. ELISA method was used to detect the production of gp120-specific antibody in rabbit serum after immunization. Results: Three codon optimized gp120 DNA vaccine could express gp120 correctly. These gp120 nucleic acid vaccine can immunize rabbits to produce HIV-1 envelope glycoprotein specific antibodies. CONCLUSION: Three codon optimized gp120 nucleic acid vaccines that confer consensus sequences of AE, BC and ThB of HIV-1 Chinese subtypes were successfully constructed and could induce HIV-1 envelope protein-specific immune responses.