文献类型：期刊文献

中文题名：基于昆虫细胞-杆状病毒表达系统的SARS-CoV-2 S1蛋白的高效表达

英文题名：High-yield production of SARS-CoV-2 S1 using insect cell-baculovirus expression vector system

作者：张如悦[1];高耀荣[2];王建超[2];曹磊[1];张艳敏[1];张炜坚[1];彭雯娟[3];谭文松[1,3];赵亮[1,3]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海杰威医药科技有限公司;[3]上海倍谙基生物科技有限公司

年份：2023

卷号：18

期号：1

起止页码：1

中文期刊名：中国病原生物学杂志

外文期刊名：Journal of Pathogen Biology

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

语种：中文

中文关键词：新型冠状病毒(SARS-CoV-2);新型冠状病毒肺炎(COVID-19);S1蛋白;昆虫细胞-杆状病毒表达载体系统

外文关键词：SARS-CoV-2;COVID-19;S1 protein;Insect cell-baculovirus expression vector system

摘要：目的 设计、构建表达新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2) S1蛋白的重组杆状病毒,建立并优化其在昆虫细胞中的生产工艺,为新型冠状病毒肺炎(COVID-19)血清学诊断方法的建立及疫苗开发奠定基础。方法 利用分子克隆技术将S1基因插入pFastBac^(TM)1质粒,获得重组质粒pFastBac-S1,经转座获得重组杆粒rbacmid-S1,转染Sf9细胞后包装获得重组杆状病毒rBV-S1,通过rBV-S1感染High Five^(TM)(Hi5)细胞表达S1蛋白,使用Western blot分析鉴定S1蛋白的表达。在此基础上,建立优化rBV-S1扩增和蛋白表达的工艺,以实现S1蛋白的高效表达。结果 通过特异性引物PCR扩增重组质粒pFastBac-S1与重组杆粒rbacmid-S1,获得大小分别为2 367 bp、4 370 bp的基因片段,表明S1基因成功克隆入pFastBac^(TM)1质粒与bacmid;采用Western blot分析细胞培养上清液,在78 ku处有特异性反应条带,证明在昆虫细胞-杆状病毒表达载体系统(Insect cell-baculovirus expression vector system, IC-BEVS)中成功表达有反应原性的S1蛋白。Sf9细胞以1.5×10^(6)cells/mL接种,MOI=0.01接毒,96 hpi可收获最大病毒滴度为(8.40±0.16)lgTCID_(50)/mL;Hi5细胞以0.8×10^(6)cells/mL接种后培养36 h,以MOI=1接毒,48 hpi可获得最大S1蛋白产量为(4.33±0.09) mg/L。结论 利用IC-BEVS成功表达了S1蛋白,并建立了优化的S1蛋白生产工艺,实现了S1蛋白的高效表达,对COVID-19临床诊断方法的建立及疫苗开发具有重要意义。
Objective To design and construct a recombinant baculovirus expressing the S1 protein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),establish and optimise its production process in insect cells, and thus providing a new option for the establishment of serological assays and the development of vaccines for COVID-19. Method The S1 gene was inserted into pFastBac^(TM)1 plasmid using molecular cloning technique to obtain the recombinant plasmid pFastBac-S1,and the recombinant rbacmid-S1 was obtained by transposition. Sf9 cells were transfected with rbacmid-S1 and then packaged to obtain recombinant baculovirus rBV-S1. High Five^(TM)(Hi5) cells were infected by the rBV-S1 to express S1 protein which was later identified using Western blotting. On this basis, the multiplicity of infection(MOI) and time of harvest(TOH) of the recombinant baculovirus amplification process were optimized, and the cell lines, MOI,time of infection(TOI) and TOH of the S1 protein expression process were optimized. Result PCR with specific primers amplified the recombinant plasmid pFastBac-S1 and the recombinant bacmid-S1,obtaining fragments of 2 367 bp and 4 370 bp respectively, indicating that the S1 gene was successfully cloned into the pFastBac^(TM)1 plasmid and bacmid. Western blotting of the cell culture supernatant showed a specific reaction band at 78 ku, demonstrating the successful expression of the reactogenic S1 protein using the insect cell-baculovirus expression vector system(IC-BEVS). For amplification of rBV-S1,the optimal process was to inoculate Sf9 cells at 1.5×10^(6) cells/mL,infect with MOI=0.01 and harvest end point at 96 hpi, thus achieving a maximum virus titer of(8.40±0.16) lgTCID_(50)/mL. For the S1 protein production, the optimal process was to inoculate Hi5 cells at 0.8×10^(6) cells/mL and culture for 36 h, infect with MOI=1 and harvest at 48 hpi, thus obtaining a maximum S1 protein yield of(4.33±0.09) mg/L. Conclusion By using IC-BEVS,we have successfully expressed the S1 protein and optimized its production process achieving high expression, which is important for the establishment of clinical diagnosis methods and the development of vaccines of COVID-19.