重组2型脊髓灰质炎病毒样颗粒的制备及免疫原性初步研究  

Preparation and immunogenicity evaluation of recombinant poliomyelitis type 2 virus-like particles

作　　者：徐洋 英志芳[2] 许琳 王剑锋[2] 刘悦越[2] 李洪艳 韩琪琪 晏巧玲 朱涛[1,3] Xu Yang;Ying Zhifang;Xu Lin;Wang Jianfeng;Liu Yueyue;Li Hongyan;Han Qiqi;Yan Qiaoling;Zhu Tao(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China;National Institutes for Food and Drug Control,WHO Collaborating Centre for Standardization and Evaluation of Biological Products,Beijing 102629,China;The Fifth Vaccine Department,Consino Biologics Inc.,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,300457 [2]中国食品药品检定研究院,WHO生物制品标准化和评价合作中心,北京102629 [3]康希诺生物股份公司疫苗五室,天津300457

出　　处：《中华微生物学和免疫学杂志》2021年第10期784-790,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项"重大新药创制"(2015ZX09102009-001)。

摘　　要：目的构建共表达P1和3CD的重组杆状病毒,利用昆虫细胞重组表达2型脊髓灰质炎病毒(poliovirus type 2,PV2)的病毒样颗粒(virus-like particle,VLP),制备PV2-VLP进行初步免疫原性研究。方法基于粉纹夜蛾细胞(High 5)密码子偏好性,将PV2的P1基因和3CD基因进行序列优化,并对P1基因进行稳定性突变,构建突变型rBac-PV2-P1s-3CD和野生型rBac-PV2-P1-3CD杆状病毒。Western blot分别检测目的蛋白表达量,离子交换层析纯化PV2-VLP,透射电镜观察VLP高级结构确定组装效率。免疫大鼠评估免疫原性。结果成功构建了可稳定表达P1s蛋白和3CD蛋白的重组杆状病毒。Western blot结果表明热稳定性突变后VLP产量相比野生型有所提高。电镜观察发现直径约30 nm的三维结构,表明VLP成功组装。动物实验结果表明,重组制备的PV2-VLP具有免疫原性,且能有效刺激产生中和抗体。结论用昆虫细胞-杆状病毒表达系统可成功制备有效的VLP类疫苗,为PV-VLP疫苗的研制提供参考。Objective To express virus-like particles of poliovirus type 2(PV2-VLP)in insect cells using a recombinant baculovirus expressing P1 and 3CD and to preliminarily evaluate its immunogenicity.Methods Based on the codon preference of High 5 cells,the sequences of P1 gene and 3CD gene of PV2 were optimized and inserted into pUC57-Amp to construct pUC57-PV2-P1 and pUC57-PV2-3CD.UC57-PV2-P1s mutant that carried P1 gene mutation affecting thermostability was then constructed.Recombinant baculovirus strains of rBac-PV2-P1s-3CD and rBac-PV2-P1-3CD(wild type)were constructed using homologous recombination.The expression of target proteins was detected by Western blot.PV2-VLP was purified by ion exchange chromatography.The structure of VLP was observed under transmission electron microscopy to evaluate the assembly efficiency.The immunogenicity of PV2-VLP was assessed in a rat model.Results The recombinant baculovirus with stable expression of P1s and 3CD proteins was successfully constructed.Western blot results showed that the yield of VLP was higher after thermostability mutation than that of the wild type.A three-dimensional structure with a diameter of about 30 nm was observed under electron microscopy,indicating that the VLP was successfully assembled.Animal experiment showed that the recombinant PV2-VLP had immunogenicity and could effectively induce the production of neutralizing antibodies.Conclusions Effective VLP vaccines could be successfully prepared using the insect cell-baculovirus expression system,which provided reference for the development of polio VLP vaccine.

关 键 词：脊髓灰质炎病毒 病毒样颗粒 昆虫细胞 重组杆状病毒 

分 类 号：R392-33[医药卫生—免疫学]