SARS-CoV-2棘突蛋白特异性多肽的筛选及初步验证  

Screening and preliminary verification of specific polypeptides of SARS-CoV-2 spike protein

作　　者：黄明柳 赖小敏 HUANG Ming-liu;LAI Xiao-min(Department of Microbiology,Zhongshan School of Medicine,Key Laboratory of Tropical Disease Control,Institute for Tuberculosis Control,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]中山大学中山医学院微生物学教研室/热带病防治研究教育部重点实验室/结核病研究所/海洋微生物功能分子广东省高校重点实验室/广东省重大传染病预防和控制技术中心,广州510080

出　　处：《中国医药生物技术》2021年第5期444-452,共9页Chinese Medicinal Biotechnology

摘　　要：目的筛选及初步验证新型冠状病毒(SARS-CoV-2)棘突蛋白(S蛋白)的特异性多肽。方法用多种生物信息学软件对SARS-CoV-2 S蛋白区别于HCoV-229E、HCoV-HKU1、HCoV-NL63、HCoV-OC43、SARS-CoV、MERS-CoV S蛋白氨基酸序列的特异性序列进行筛选并预测其理化性质、空间结构。根据筛选、预测结果,将SARS-CoV-2 S蛋白特异性序列进行化学合成或人工表达。运用酶联免疫吸附(ELISA)实验验证这些基于生物信息学筛选出的特异性多肽的抗原性、免疫原性以及证明抗原性最强的特异性多肽在实际应用中检测COVID-19抗体阳性患者血清的可行性和特异性。结果选出5条具有抗原性与免疫原性的多肽,S2、S3、S6、S7,以及S6与S7用linker连接大肠埃希菌表达的小分子融合蛋白(S6-Linker-S7);S6-Linker-S7与SARS-CoV-2 S蛋白对比检测同一批普通呼吸系统疾病患者血清IgG抗体平均检测值OD_(450)nm分别为(0.70±0.34)、(1.33±0.54),检测值差异具有统计学意义(P<0.001),对比检测COVID-19抗体阳性患者血清IgG抗体检测值OD_(450)nm分别为(2.20±1.95)、(2.35±0.57),检测值差异不具有统计学意义(P>0.05);S6-Linker-S7在检测COVID-19抗体阳性患者时,血清IgG抗体检测值OD_(450)nm均值比检测普通呼吸疾病患者OD_(450)nm均值高3倍,差异具有统计学意义(P<0.001);SARS-CoV-2 S蛋白与HCoV-HKU1、HCoV-OC43、SARS-CoV S蛋白兔多抗发生免疫反应;S6-Linker-S7与抗HCoV-OC43、SARS-CoV S蛋白兔多抗发生免疫反应。结论实验证明了针对SARS-CoV-2 S蛋白,基于生物信息学技术设计合成的区别于HCoV-229E、HCoV-HKU1、HCoV-NL63、HCoV-OC43、SARS-CoV、MERS-CoV的特异性多肽具有抗原性和免疫原性,S6-Linker-S7能在实际应用中检测COVID-19抗体阳性患者血清,并且相较于SARS-CoV-2 S蛋白具有更高的特异性。Objective To screen and preliminary identify the specific peptides of SARS-CoV-2 spike protein(S protein).Methods A variety of bioinformatics softwares were used to screen the specific amino acid sequences of SARS-CoV-2 S protein different from HCoV-229E,HCoV-HKU1,HCoV-NL63,HCoV-OC43,SARS-CoV and MERS-CoV S protein,and predict its physicochemical properties and spatial structure.According to the screening and prediction results,the specific sequence of SARS-CoV-2 S protein was chemically synthesized or artificially expressed.Enzyme linked immunosorbent assay(ELISA)was used to verify the antigenicity and immunogenicity of these specific peptides screened based on bioinformatics,and to prove the feasibility and specificity of the strongest antigenic peptide in detecting the serum of patients with COVID-19 positive antibodies in practical application.Results Five peptides with antigenicity and immunogenicity,S2,S3,S6,S7,and S6-Linker-S7 were screened.Compared the detection of S6-Linker-S7 to SARS-CoV-2 S protein,the average OD450 nm values of serum IgG antibodies in patients with common respiratory diseases were(0.70±0.34)and(1.33±0.54)respectively,and the difference was statistically significant(P<0.001).The values of serum IgG antibody in patients with COVID-19 positive antibodies were(2.20±1.95)and(2.35±0.57)respectively,but the difference of OD450 nm values was not statistically significant(P>0.05).When S6-Linker-S7 was used to detect COVID-19 antibodies in positive patients,the mean OD450 nm value of serum IgG antibodies was 3 times higher than that of patients with common respiratory diseases and the difference was statistically significant(P<0.001).SARS-CoV-2 S protein reacted with rabbit polyclonal antibodies targeted to HCoV-HKU1,HCoV-OC43 and SARS-CoV S protein and S6-Linker-S7 reacted with rabbit polyclonal antibodies against HCoV-OC43 and SARS-CoV S protein.Conclusion It is proved in experiments that the specific polypeptides designed and synthesized for SARS-CoV-2 spike protein based on bioinformati

关 键 词：新型冠状病毒 棘突蛋白 特异性多肽 抗原性 免疫原性 ELISA 

分 类 号：R446.6[医药卫生—诊断学]