猴痘病毒A29L-A35R-M1R融合蛋白的原核表达、纯化及免疫原性初步分析

Prokaryotic Expression,Purification and Preliminary Immunogenicity Analyses of the A29L-A35R-M1R Fusion Protein of the Monkeypox Virus

猴痘病毒(Monkeypox virus,MPXV)是一种能够感染人和动物的痘病毒,已对人类公共卫生安全构成巨大威胁,安全高效的疫苗能够有效预防和控制病毒的传播。本研究选取MPXV A29L、A35R和M1R氨基酸序列通过柔性Linker(GGGGS)连接设计融合蛋白,并根据大肠杆菌密码子偏嗜性对其密码子优化后合成全基因,插入pET-28a载体,构建pET28a-A29L-A35R-M1R重组质粒。转化至E.coli BL21(DE3)感受态细胞中诱导表达,经SDSPAGE和Western blot进行鉴定,并确定最佳诱导条件。在此基础上,对融合蛋白rA29L-A35R-M1R进行Ni-NTA亲和层析柱纯化后,免疫小鼠。通过ELISA方法检测抗MPXV A29L、A35R和M1R特异性IgG和细胞因子(IFN-γ、IL-2和IL-4)水平,评价融合蛋白免疫原性。实验结果显示:重组质粒pET-28a-A29L-A35R-M1R在E.coli BL21(DE3)感受态细胞中成功表达,最佳表达条件为37℃、0.25 mmol/L IPTG诱导5 h;将纯化的融合蛋白rA29LA35R-M1R免疫小鼠3次后,能够产生抗A29L、A35R和M1R特异性IgG,其中anti-rA29L-A35R-M1R IgG水平最高,且融合蛋白rA29L-A35R-M1R能够刺激机体产生高水平IFN-γ,IL-2和IL-4,与对照组差异均具有统计学意义(P<0.01)。由此表明,融合蛋白rA29L-A35R-M1R具有良好的免疫原性。为猴痘病毒新型疫苗和治疗性生物制剂的研发提供理论依据。

The monkeypox virus(MPXV)is a poxvirus that can infect humans and animals.The MPXV poses a significant threat to public-health security.A safe and efficacious vaccine is needed to prevent and control the spread of the MPXV.The amino-acid sequences of A29L,A35R and M1R of the MPXV were selected to design fusion proteins using a flexible peptide(GGGGS)linkage.The whole gene was synthesized after codon optimization according to Escherichia coli codon bias,inserted into the pET-28a vector,and then we constructed the pET28a-A29L-A35R-M1R recombinant plasmid.Expression was induced by transformation into E.coli BL21(DE3)receptor cells,characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting,and the optimal induction conditions were determined.On this basis,the fusion protein rA29L-A35R-M1R was purified by Ni-NTA affinity chromatography and immunized to mice.The immunogenicity of fusion proteins was evaluated by measuring the levels of anti-MPXV A29L,A35R and M1R-specific immunoglobulin(Ig)G and cytokines(interferon(IFN)-γ,interleukin(IL)-2 and(IL)-4)by enzyme-linked immunosorbent assays.The recombinant plasmid pET-28a-A29L-A35R-M1R was expressed in E.coli BL21(DE3)receptor cells.The optimal conditions for expression were use of IPTG(0.25 mmol/L)at 37°C for 5 h.After immunizing mice thrice with the purified fusion proteins rA29L-A35R-M1R,mice could produce anti-A29L,A35R and M1R-specific IgG.The level of anti-rA29L-A35R-M1R IgG was the highest.The fusion protein rA29L-A35R-M1R could stimulate the body to produce high levels of IFN-γ,IL-2 and IL-4,and the differences in levels compared with those in the control group were all highly significant(P<0.01).These data indicate that the fusion protein rA29L-A35R-M1R had good immunogenicity.Our results provide a theoretical basis for the development of novel vaccines and therapeutic biological agents for the MPXV.