目的 通过随机突变策略，模拟SARS-CoV-2的变异进化，构建RBD突变抗原库，使用不同血清流式筛选具备产生较强交叉结合活性抗血清潜力的候选抗原，分析比较候选抗原的抗原性和免疫原性的特征和差异，探讨随机突变产生的新位点对抗原的影响。

方法 利用易错耐热DNA聚合酶对Delta、Omicron BA.1、BF.7三种毒株的RBD+CTD1序列进行扩增，构建突变抗原库；将突变抗原通过同源重组的方法构建至带有穿膜区的膜抗原表达载体上；通过流式细胞术，用康复期患者血清检测突变库抗原，初步筛选出具备交叉识别能力的候选抗原；构建候选抗原分泌型表达载体，表达纯化候选抗原并鉴定其分子量、纯度等；将候选抗原按照0天和14天的免疫程序免疫小鼠，28天时采血，用ELISA方法检测免疫血清与6种VOC蛋白（WT\Beta\Delta\Omicron BA.1\BF.7\XBB.1.5）的交叉识别能力；并通过ELISA、MST、ACE2 binding等实验检测候选抗原的抗原性；通过点突变的方法，增减候选抗原上的由于突变引入的糖基化位点，探索糖基化位点对候选抗原的抗原性的影响。

结果 ①构建了一个包含364条随机突变抗原序列的突变库；②应用膜抗原筛选策略，初步筛选表达了6条具有交叉识别能力的候选抗原（Delta组的S3-2-B9、S3-B7，BA.1组的S5-3-C6和S5-3-D6-C2，BF.7组的S7-7-C7、S7-1-H5）；③免疫动物实验表明，S3-2-B9、S3-B7、S7-7-C7、S7-1-H5等候选抗原能诱导产生具有较强交叉结合活性的抗血清（可以交叉识别WT\Beta\Delta\Omicron BA.1\BF.7\XBB.1.5等VOC蛋白）；④S3-2-B9、S7-1-H5分别含有A372T、N542T等糖基化位点，当这些糖基化位点与序列上的其它突变位点组合时，会提升候选抗原的抗原性及免疫原性。

结论 ①基于随机突变的叠加突变可以有效提高突变抗原库的容量；②流式细胞术检测HEK293T细胞膜展示突变抗原库的方法，能够初步达到高通量筛选具有较强交叉识别能力的候选抗原的目的；③A372T、N542T等糖基化位点组合其他突变位点，可提高候选抗原的抗原性及免疫原性。

Objective A library of RBD mutant antigens was constructed by a random mutation strategy, simulating the mutational evolution of SARS-CoV-2. Candidate antigens with the potential to generate strong cross-binding activity antisera were screened using different serum flow-throughs to analyze and compare the characteristics and differences in antigenicity and immunogenicity of the candidate antigens, and to explore the effect of the new sites generated by the random mutations on the antigens.

Methods The RBD+CTD1 sequences of Delta, Omicron BA.1 and BF.7 strains were amplified by error-prone heat-resistant DNA polymerase to construct a library of mutant antigens; the mutant antigens were constructed by homologous recombination into membrane antigen expression vectors with a membrane-penetrating region; the antigens in the library were detected with serum of convalescent patients by flow cytometry to preliminarily screen out the candidate antigens with the ability of cross-recognition. Candidate antigens with cross-recognition ability are preliminarily screened out by flow cytometry; Candidate antigens are constructed into secretory expression vectors, and the molecular weights and purities of the candidate antigens are identified after expression and purification; The candidate antigens are immunized into mice according to the immunization procedures of 0 and 14 days, and blood is collected at 28 days, and the immunized sera are tested for the association of 6 VOC proteins (WT\Beta\Delta\Omicron BA.1\BF.7\) with the immunized antigens using the ELISA method. XBB.1.5); and the antigenicity of the candidate antigens was tested by ELISA, MST, and ACE2 binding; the effect of glycosylation sites on the antigenicity of the candidate antigens was explored by adding or subtracting glycosylation sites on the candidate antigens that were introduced due to mutations by point mutation.

Results ① a mutation library containing 364 randomly mutated antigen sequences was constructed; ②applying the membrane antigen screening strategy, six candidate antigens with the ability of inducing strong cross-recognition of antisera were preliminarily screened and expressed (S3-2-B9 and S3-B7 in the Delta group, S5-3-C6 and S5-3-D6-C2 in the BA.1 group, S7-7-C7 in the BF.7 group, S7-1 -H5); ③ Immunization animal experiments showed that candidate antigens such as S3-2-B9, S3-B7, S7-7-C7, and S7-1-H5 induced the production of antisera with strong cross-binding activity (which can cross-recognize the proteins of the virulent strains such as WT\Beta\Delta\Omicron BA.1\BF.7\XBB.1.5); ④ S3-2-B9, S7-1-H5 contain glycosylation sites such as A372T, N542T, etc., respectively, which, when combined with other mutation sites on the sequence, produce an enhancement of antigenicity and immunogenicity of the candidate antigen.

Conclusions  ① superimposed mutations based on random mutations can effectively increase the capacity of the mutant antigen library; ②the method of flow cytometry for detecting the membrane-displayed mutant antigen library of HEK293T cells can initially achieve the purpose of high-throughput screening of candidate antigens with strong cross-recognition ability; ③the combination of other mutation sites such as glycosylation sites A372T, N542T, and so on, can improve the antigenicity and immunogenicity of the candidate antigens.