优化SARS-CoV-2 S-EABR在293T细胞中的分泌性表达  

Optimizing the secretory expression of SARS-CoV-2 S-EABR in 293T cells

作　　者：姜苏芮 毛彤瑶 张鹏 段招军 Jiang Surui;Mao Tongyao;Zhang Peng;Duan Zhaojun(School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;NHC Key Laboratory of Medical Virology and Viral Disease,National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]甘肃中医药大学公共卫生学院,兰州730000 [2]中国疾病预防控制中心病毒病预防控制所,传染病溯源预警与智能决策全国重点实验室,国家卫生健康委员会医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2024年第5期489-496,共8页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划(2022YFC2304302)。

摘　　要：目的通过优化启动子、终止子、信号肽及胞内序列末端氨基酸来增加SARS-CoV-2 S-EABR蛋白在293T细胞系中的分泌性表达。方法首先构建四种含有不同元件(启动子和终止子)组合的PCDNA3.1(-)真核载体质粒(Mb、MS、Ab、AS),插入按照人密码子优化的S-EABR-1目的序列,瞬时转染293T细胞,48 h后收取细胞培养上清液和细胞裂解液,通过Western Blot和ELISA检测上清S蛋白的表达水平。然后选择表达最好的元件组合载体,插入S-EABR-1和S-EABR-2(于S-EABR-1尾部添加4个氨基酸——HSLP)目的序列,比较上清S蛋白的表达水平。最后基于上述表达效果最好的元件组合载体及插入目的序列,构建替换SARS-CoV-2 S蛋白原始信号肽的5种载体质粒(tPA,AZ,IFNα2,HSA,GLUC),比较上清S蛋白表达水平。同时利用计算机模拟SRP54亚基与信号肽核酸序列的分子对接,将对接分值(Docking Score)作为对接评价标准,预测两者的结合情况。结果在293T细胞中,Ab组合的载体分泌表达S-EABR水平最高,产量相较于Mb增加了125%。基于Ab组合载体,S-EABR-2序列表达分泌S-EABR的水平较S-EABR-1增加了约50%,进一步替换成HSA信号肽后的S-EABR表达分泌水平较S蛋白原始信号肽增加了约83%。此外,计算机模拟结果显示HSA与SRP54亚基的对接分值最高,为1505.861。结论通过真核表达元件(启动子、终止子和信号肽)及胞内序列的优化可进一步提高经密码子优化后的S-EABR在293T细胞中的分泌性表达。信号肽与SRP54亚基亲和力的计算模拟对接分值与S-EABR分泌表达水平基本吻合的结果也为后续提高目的基因分泌性表达的信号肽筛选提供了一种设计思路和筛选策略。ObjectiveTo increase secretory expression of SARS-CoV-2 S-EABR protein in 293T cell line by optimizing promoter,PolyA signals,signal peptide and terminal amino acids of intracellular sequences.MethodsFirst,four PCDNA3.1(-)eukaryotic vector plasmids(Mb,MS,Ab,AS)containing different combinations of elements(promoter and PolyA signals)were constructed,and the S-EABR-1 target sequence optimized according to human codons was inserted.293T-cells were transiently transfected.After 48 hours,cell culture supernatants and cell lysates were collected,and the expression level of S protein in supernatant was detected by Western blotting and ELISA.Then,the vector with the best expression element combination was selected,and the target sequences of S-EABR-1 and S-EABR-2(4 amino acids-HSLP were added to the tail of S-EABR-1)were inserted to compare the expression level of S protein in the supernatant.Finally,based on the combination of the above elements with the best expression effect and the insertion of the target sequence,five vector plasmids(tPA,AZ,IFNα2,HSA,GLUC)were constructed to replace the original signal peptide of SARS-CoV-2 S protein,and the expression level of S protein in the supernatant was compared.At the same time,a computer was used to simulate the molecular docking of the SRP54 subunit and the signal peptide nucleic acid sequence,and the Docking Score was used as the docking evaluation criterion to predict the binding of the two.ResultsIn 293T cells,the Ab combination vector secreted the highest level of S-EABR,and the yield increased by 125%compared with Mb.Based on the Ab combination vector,the level of S-EABR-2 sequence expression and secretion of S-EABR increased by about 50%compared with S-EABR-1.After further replacement with the HSA signal peptide,the level of S-EABR expression and secretion increased by about 83%compared with the original signal peptide of the S protein.In addition,computer simulation result showed that the docking score between HSA and SRP54 subunit was the highest,at 1505.861.Concl

关 键 词：新型冠状病毒 S-EABR 启动子 终止子 信号肽 信号识别颗粒 

分 类 号：R373[医药卫生—病原生物学]