【摘要】 目的 从mRNA序列优化和体外转录(IVT)系统优化角度，设计2条基于血凝素(HA)基因的抗H1N1亚型流感病毒mRNA疫苗，分别连接到3种体外转录系统(含不同UTRs序列)中进行候选疫苗抗原的表达及鉴定，并确定候选疫苗所使用的最佳体外转录系统。方法 确保抗原基因(HA)氨基酸序列不变，以2种优化策略对HA基因的密码子进行优化，分别命名为JLH1HA和JLHA,并分别克隆至骨架载体pGEM-T7-Hα(UTRs来自人源α球蛋白)、pGEM-T7-Mα(UTRs来自鼠源α球蛋白)、pGEM-n3(UTRs来自人源β球蛋白)上，通过线性化处理、体外转录、纯化及Cap1加帽处理，获得功能性mRNA,转染A549细胞后通过Western blot和间接免疫荧光试验(IFA)鉴定目的蛋白的表达。结果 成功构建6组mRNA候选疫苗且均能表达抗原蛋白，以pGEM-T7-Hα为骨架载体构建的体外转录系统蛋白表达量较高。结论 抗原的设计具有可行性，筛选出的最优体外转录系统为以pGEM-T7-Hα为骨架载体构建的体外转录系统。密码子优化对蛋白表达量的影响并不显著，可能在动物免疫效果上能够体现。更多还原

【Abstract】 Objective From the perspective of mRNA sequence optimization and in vitro transcription(IVT) system optimization, two mRNA vaccines against H1N1 subtype influenza virus based on hemagglutinin(HA) gene were designed and linked to three in vitro transcription systems(including different UTRs sequences) respectively for the expression and identification of candidate vaccine antigens, and the optimal in vitro transcription system used for candidate vaccines was determined. Methods To ensure that the amino acid sequence of antigen gene(HA) remained unchanged, two optimization strategies were used to optimize the codon of HA gene, named JLH1HA and JLHA,respectively. They were cloned into the skeleton vectors pGEM-T7-Hα(UTRs from human α globulin),pGEM-T7-Mα(UTRs from mouse α globulin) and pGEM-n3(UTRs from human β globulin),respectively. Functional mRNA was obtained by linearization, in vitro transcription, purification and Cap1 capping treatment. A549 cells were transfected with Western blot and indirect immunofluorescence assay(IFA) to identify the expression of target protein. Results Six groups of mRNA candidate vaccines were constructed successfully, and all of them could express antigen proteins successfully. Among them, the protein expression of the transcription system constructed with pGEM-T7-Hα as the skeleton vector was the highest. Compared with pGEM-n3,the difference was signficant(P<0.01). Conclusion The design of antigen was feasible, and the optimal in vitro transcription system was pGEM-T7-Hα skeleton carrier. The effect of codon optimization on protein expression is not significant, which may be reflected in animal immune effect.更多还原