重组SARS-CoV-2 Omicron变异株S1蛋白的表达及免疫原性评价  

Expression of the S1 Protein for Recombinant SARS-CoV-2 Omicron Variants and Its Immunogenicity Evaluation

作　　者：卢慧敏 王梓豪 边成 王晓辉[1] 李吉翠 马绍辉[1] 褚嘉祐[1] 杨昭庆[1] LU Huimin;WANG Zihao;BIAN Cheng(Laboratory of Medical Genetics,Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Yunnan 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所医学遗传学研究室,昆明650118 [2]昆明医科大学,650500

出　　处：《医学研究杂志》2024年第8期126-131,共6页Journal of Medical Research

基　　金：云南省高层次卫生健康技术人才培养专项(L-2018003);中国医学科学院医学与健康科技创新工程项目创新疫苗关键技术研究项目(2021-I2M-1-043)。

摘　　要：目的构建表达新型冠状病毒(SARS-CoV-2)Omicron BA.1变异株S1蛋白的真核表达载体,在CHO-K1细胞中进行表达,评价其免疫原性,并对佐剂和抗原剂量进行研究。方法构建重组质粒UCOE-Omi-S1,转染至CHO-K1工程细胞中进行表达和纯化,通过SDS-PAGE和Western blot法实验鉴定重组S1蛋白。将BALB/c小鼠随机分为12组,即PBS组,无佐剂组(高、中、低剂量组),铝盐佐剂对照组,MF59佐剂对照组,铝盐佐剂实验组(高、中、低剂量组),MF59佐剂实验组(高、中、低剂量组),将按照分组要求配比的溶液经小鼠肌内注射3次,间隔14天,每2周尾静脉采血,末次免疫30天后取血分离血清,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清抗体水平,并和SARS-CoV-2原型株的假病毒和SARS-CoV-2 Omicron BA.1变异株的假病毒进行假病毒中和实验。结果目的蛋白在CHO-K1工程细胞表达后可分泌到培养上清中,在相对分子质量约为70kDa处可见1条特异性蛋白表达条带,经Western blot法鉴定为Omi-S1蛋白。铝盐佐剂组和MF59佐剂组免疫诱导效果优于无佐剂组,铝盐佐剂组和MF59佐剂组免疫诱导效果相差不大,但MF59佐剂起效快;高、中、低剂量组的免疫诱导效果在第8周相差不大,但高剂量组起效快。假病毒中和实验表明,MF59佐剂实验组针对Omicron变异株的中和抗体水平高于铝盐佐剂组。结论本研究所构建的Omicron S1重组蛋白免疫原性良好,在小鼠体内产生了良好的体液免疫应答,并诱导高水平的对抗SARS-CoV-2假病毒的中和抗体,对佐剂和抗原剂量初步研究,结果显示,10μg以下抗原剂量搭配MF59佐剂可获得较好的免疫效果。本研究为SARS-CoV-2变异株的重组蛋白疫苗的研制提供了实验基础。Objective To construct a eukaryotic expression vector for the SARS-CoV-2 Omicron BA.1mutant S1 protein,expressed in CHO-K1 cells to evaluate its immunogenicity,and investigate the adjuvant and antigen dosages.Methods The recombinant plasmid UCOE-Omi-S1 was constructed and transfected into CHO-K1 engineering cells for expression and purification.The recombinant S1 protein was confirmed using SDS-PAGE and Western blot.BALB/c mice were randomly divided into 12groups:PBS group,no adjuvant group(high,medium and low-dose groups),aluminum salt adjuvant control group,MF59 adjuvant control group,aluminum salt adjuvant experimental group(high,medium and low-dose groups),MF59 adjuvant experimental group(high,medium and low-dose groups).The solution in accordance with the required proportion of the group was injected intramuscular into the mice 3 times,the interval of 14days,and the blood was collected from the tail vein every 2 weeks,and the serum was separated 30days after the last immunization.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum antibody levels and to neutralize pseudoviruses with SARS-CoV-2 prototype and SARS-CoV-2 Omicron BA.1 variant strains.Results After the expression of the target protein in CHO-K1 engineering cells,the expressed protein was observed at a relative molecular mass of approximately 70kDa and was identified as Omi-S1 protein by Western blot.The immune induction effect of the aluminum salt adjuvant group and MF59 adjuvant group was better than that of the no adjuvant group.There was no significant difference between the aluminum salt adjuvant group and the MF59 adjuvant group,but the MF59 adjuvant took effect more quickly.The immune induction effect of the high,middle,and low-dose groups was similar at the 8th week,but the effect of the high-dose group was faster.Pseudovirus neutralization experiments showed that MF59 adjuvant experimental group exhibited higher levels of neutralizing antibodies against the Omicron variants than the aluminum salt adjuvant group.Conclusion Thi

关 键 词：SARS-CoV-2 重组蛋白疫苗 佐剂 假病毒 中和抗体 

分 类 号：R392[医药卫生—免疫学]