严重急性呼吸综合征冠状病毒2受体结合域蛋白的分泌表达及其免疫原性分析  

Secretory expression and immunogenicity analysis of SARS-CoV-2 RBD protein

作　　者：童光伟 赵大鹏 刘丽 高宁 尹金良 卢颖林 TONG Guangwei;ZHAO Dapeng;LIU Li;GAO Ning;YIN Jinliang;LU Yinglin(School of Pharmacy,Wuwei Occupational College,Wuwei 733000,Gansu Province,China)

机构地区：[1]武威职业学院药学院,甘肃武威733000 [2]长春生物制品研究所有限责任公司疫苗研究室,吉林长春130012 [3]深圳鑫泰康生物科技有限公司,广东深圳518000 [4]国药中国生物技术股份有限公司,北京130012 [5]广东省科学院生物与医学工程研究所,广东广州510316

出　　处：《中国生物制品学杂志》2024年第8期911-916,931,共7页Chinese Journal of Biologicals

基　　金：中央引导地方科技发展资金(YDZX20202200004719-1);吉林省科技发展计划项目(20200301048RQ).

摘　　要：目的原核可溶性表达严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARSCoV-2)受体结合域(receptor binding domain,RBD)蛋白,纯化后免疫小鼠,检测其免疫原性,为SARS-CoV-2候选疫苗的制备提供新的思路。方法扩增SARS-CoV-2 RBD蛋白基因片段,酶切后与pNCMO2表达载体连接,构建重组表达质粒pNC-RBD。将重组原核表达质粒转化入短小芽孢杆菌感受态细胞,扩大培养并降温诱导蛋白表达。利用亲和层析纯化目的蛋白,经氢氧化铝佐剂吸附后经背部皮下免疫雌性BALB/c小鼠,以仅免疫铝佐剂为对照,每组10只。ELISA法检测体液免疫效果,体外增殖试验测定细胞免疫效果,ELISA法测定多种细胞因子的体外分泌,流式细胞术测定细胞分型。结果重组表达质粒pNC-RBD经菌落PCR及测序证明构建正确。重组蛋白相对分子质量约22000,可分泌表达。纯化后重组蛋白纯度达95%以上。免疫小鼠后血清结合抗体和中和抗体效价分别可达1∶10000和1:750左右,均显著高于佐剂组(t分别为2.845和2.528,P均<0.01);体外可刺激淋巴细胞增殖,并促进IL-1β及IFNγ的分泌;细胞分型试验结果表明促进了CD4~+和CD8~+T细胞淋巴细胞增殖。结论成功可溶性表达了SARS-CoV-2 RBD蛋白,其免疫原性良好,为以RBD蛋白为基础研制SARS-CoV-2基因工程疫苗奠定了基础。Objective To express the receptor binding domain(RBD)protein of severe acute respiratory symptom coronavi-rus 2(SARS-CoV-2)in soluble form by prokaryotic system and evaluate its immunogenicity by immunizing mice with the purified protein,so as to provide a new idea for the preparation of SARS-CoV-2 candidate vaccine.Methods SARS-CoV-2 RBD gene fragment was amplified and ligated with pNCMO2 vector after enzyme digestion.The obtained recombinant pro-karyotic expression plasmid pNC-RBD was transformed into Bacillus choshinensis competent cells,which were subjected to expanded culture and induced by lowering temperature.The target protein was purified by affinity chromatography,adsorbed by aluminum hydroxide adjuvant,and then the female BALB/c mice were immunized subcutaneously in the back using the protein,10 mice for each group,with the aluminum adjuvant-free protein as control.The humoral immunity was measured by ELISA,while the cellular immune effect by proliferation assay in vitro.The secretion of various cytokines in vitro was measured by ELISA,and the cell typing was determined by flow cytometry.Results The recombinant expression plasmid pNC-RBD was constructed correctly as identified by colony PCR and sequencing.The recombinant protein had a relative molecular mass of about 22000,which could be expressed and secreted,and the purity of recombinant protein after purifi-cation was over 95%.The titers of binding antibody and neutralizing antibody in serum of the immunized mice were about 1:10000 and 1:750,respectively,which were significantly higher than those in adjuvant group(t=2.845 and 2.528,respectively,each P<0.O1).The recombinant protein stimulated lymphocyte proliferation and promoted the secretion of IL-1βand IFN in vitro.The results of cell typing test showed that the lymphocyte proliferation of both CD4 and CD8*T cells was promoted.Conclusion The RBD protein of SARS-CoV-2 was successfully expressed in soluble form with good immunogenicity,which lays a foundation for the development of SARS-CoV-2 genet

关 键 词：严重急性呼吸综合征冠状病毒2 受体结合域蛋白 可溶性表达 免疫原性 

分 类 号：R373.2[医药卫生—病原生物学]