(pfs25)_(2)-CSP基因在毕赤酵母中的分泌表达及免疫原性分析  

The secretory expression of the (pfs25)_(2)-CSP gene in Pichia pastoris and analysis of the protein immunogenicity

作　　者：王爱秀 李宣钟 刘宇阳 李刚 陈勇 WANG Aixiu;LI Xuanzhong;LIU Yuyang;LI Gang;CHEN Yong(The Quality Control Department in Lanzhou Biotechnology Development Co.,Ltd.,Lanzhou 730046,Gansu Province,China;不详)

机构地区：[1]兰州生物技术开发有限公司质量检定室,甘肃兰州730046 [2]武汉生物制品研究所有限责任公司轮状病毒疫苗室,湖北武汉430200 [3]兰州生物制品研究所有限责任公司第四研究室室甘肃省疫苗技术创新中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第6期6-13,共8页Progress In Microbiology and Immunology

摘　　要：目的用毕赤酵母(Pichia pastoris)系统表达(pfs25)_(2)-CSP融合蛋白并对其免疫原性进行初步分析,为疟疾疫苗的开发提供参考。方法构建(pfs25)_(2)-CSP基因重组表达质粒后电转酵母,筛选高表达重组酵母菌株进行发酵培养和蛋白纯化,并对纯化的(pfs25)_(2)-CSP融合蛋白的免疫原性进行初步分析。结果(pfs25)_(2)-CSP基因重组表达质粒构建成功,电转后获得了(pfs25)_(2)-CSP基因重组酵母菌,目的蛋白的表达形式主要为分泌表达,分泌表达的(pfs25)_(2)-CSP融合蛋白含量约为菌内可溶性表达的15倍。经发酵罐培养和蛋白纯化获得约100%纯度的(pfs25)_(2)-CSP融合蛋白,免疫原性研究结果表明,(pfs25)_(2)-CSP融合蛋白诱导产生的抗体中,针对(pfs25)_(2)蛋白的抗体水平较高,且维持时间长,3次免疫后1周的抗-pfs25抗体效价为4×10^(2)~1×10^(4),3个月的抗-pfs25抗体效价为1×10^(4)~8×10^(4),而针对CSP蛋白的抗体水平较低,3次免疫后1周的抗-CSP抗体效价为2×10^(2)~8×10^(2),3个月的抗-CSP抗体效价未见增高。结论实现了(pfs25)_(2)-CSP基因在毕赤酵母中的分泌表达,并得到了(pfs25)_(2)-CSP融合蛋白的初步免疫原性数据,为疟疾疫苗的研发奠定了基础。Objective To express(pfs25)_(2)-CSP fusion protein in Pichia pastoris system and preliminarily analyse its immunogenicity, so as to provide reference for the development of malaria vaccine. Methods The recombinant expression plasmid of(pfs25)_(2)-CSP gene was constructed and transformed into yeast by electroporation. The recombinant yeast strains with high protein expression were screened, fermented, followed by protein purification. The immunogenicity of the purified(pfs25)_(2)-CSP fusion protein was preliminarily analysed. Results The recombinant plasmid of(pfs25)_(2)-CSP gene was successfully constructed, and the recombinant yeast bearing(pfs25)_(2)-CSP gene was obtained after electric transfer. The expression form of the target protein was mainly secreted. The amount of secreted(pfs25)_(2)-CSP fusion protein was about 15 times as many as soluble expression in the bacteria.The(pfs25)_(2)-CSP fusion protein was obtained with about 100% purity by fermenter culture and protein purification. The results of immunogenicity study showed that the antibody levels induced by the(pfs25)_(2) protein in(pfs25)_(2)-CSP fusion protein were higher and maintained for a long time, with the titers of 4×10^(2)-1×10^(4) at 1 week after the third immunization, and 1×10^(4)-8×10^(4) at 3 months after the third immunization. While antibody levels induced by CSP protein were lower, with the titer of 2×10^(2)-8×10^(2) at 1 week after the third immunization, and there was no increase in the titer of anti-CSP antibody at 3 months after the third immunization. Conclusion The secretory expression of(pfs25)_(2)-CSP gene in Pichia pastoris was realized, and the preliminary immunogenicity results were obtained, which laid the foundation for the development of malaria vaccine.

关 键 词：(pfs25)_(2)-CSP 融合基因 毕赤酵母 分泌表达 免疫原性 

分 类 号：R392[医药卫生—免疫学]