摘要
根据CHO细胞密码子偏好性进行SARS-CoV-2 Beta变异株S1基因的优化与合成,并将其克隆至pSN载体,构建重组表达质粒pSN-Beta-S1,转染CHO细胞,获得重组蛋白Beta-S1,通过SDS-PAGE和 Western blot方法鉴定重组蛋白Beta-S1的表达,对培养液上清进行亲和层析纯化.将纯化后的重组蛋白Beta-S1联合氢氧化铝佐剂免疫BALB/c小鼠,检测血清中特异性IgG抗体及其亚型,评价针对SARS-CoV-2不同变异株的交叉中和抗体活性.结果显示,成功构建pSN-Beta-S1重组质粒,经CHO细胞表达系统表达了重组蛋白Beta-S1,重组蛋白条带单一,大小约为120 kDa,纯度可达85％以上,可与SARS-CoV-2 RBD蛋白多克隆抗体和Strep标签单克隆抗体特异性结合.S1重组蛋白对BALB/c小鼠具有良好的免疫原性,第3次免疫后14 d,针对RBD和S1蛋白的特异性IgG抗体效价平均可达1∶66 260和1∶133 120,抗体亚型偏向于IgG1,针对SARS-CoV-2野生株,Beta、Delta、Omicron BA1和Omicron BA2变异株的中和抗体分别为1∶629、1∶1 720、1∶374、1∶77和1∶101.本研究利用CHO细胞表达系统成功制备了 SARS-CoV-2 Beta变异株S1重组蛋白,免疫BALB/c小鼠后获得了良好的免疫原性和针对不同变异株的交叉中和抗体活性,为广谱SARS-CoV-2疫苗的研究提供了一定的借鉴意义.
Abstract
The codon sequence of the S1 protein of the SARS-CoV-2 Beta variant was optimized ac-cording to the preference of CHO cells and cloned into pSN expression vector to construct the re-combinant plasmid pSN-Beta-sl.Recombinant protein was expressed in CHO cells,identified using SDS-PAGE and Western blot,and purified through affinity chromatography.BALB/c mice were immunized with purified recombinant protein Beta-S1 combined with aluminum hydroxide adju-vant.Specific IgG antibody and its subtypes in serum and the cross-neutralization antibody activity against SARS-CoV-2 variants were evaluated.The results showed that the recombinant plasmid pSN-Beta-S1 was successfully constructed,and the recombinant protein Beta-S1 was produced u-sing the CHO cell expression system.The purified recombinant protein had a single band of about 120 kDa with the purity exceeding 85％and can bind to RBD pAb and strep-tag mAb.The recom-binant S1 protein showed good immunogenicity in BALB/c mice.The titers of specific IgG antibodies against RBD protein and S1 protein reached 1∶66 260 and 1∶133 120 on average 21 d after the third immunization,the antibody subtypes were mainly inclined to IgG1.Serum neutrali-zing antibody titer was 1∶629 for wild type,1∶1 720 for Beta,1∶374 for Delta,1∶77 for Omi-cron BA1 and 1∶101 for Omicron BA2.In this study,S1 recombinant protein of SARS-CoV-2 Beta variant was successfully expressed in CHO cell expression system and produced good immunoge-nicity and cross-neutralizing activity in BALB/c mice.These results provide a reference for the fur-ther development of broad-spectrum SARS-CoV-2 vaccine.
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