目的·筛选对新冠病毒（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）具有中和能力的人鼠嵌合抗体，并分析其轻链上的关键氨基酸位点变化。方法·以SARS-CoV-2刺突蛋白作为免疫原，免疫抗体重链基因人源化的小鼠，通过酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）和假病毒中和实验检测小鼠血清抗体水平，应用流式细胞术分选小鼠脾脏和淋巴结中的浆细胞和生发中心（germinal center，GC）B细胞并分析各类B细胞比例。提取细胞的RNA构建鼠源抗体轻链基因文库，与固定的人源抗体重链基因配对表达抗体，并采用ELISA筛选高亲和力嵌合抗体。与鼠源胚系抗体轻链基因比对，分析高亲和力嵌合抗体轻链的氨基酸变化位点。分别构建并表达轻链单个氨基酸位点改变的嵌合抗体，通过ELISA和假病毒中和实验检测其亲和力及中和能力；比较上述位点改变前后嵌合抗体的亲和力及中和能力，并分析具有关键作用的氨基酸位点变化。结果·与未免疫小鼠相比，免疫后小鼠血清中针对SARS-CoV-2的特异性抗体的滴度增加，脾脏和淋巴结中的浆细胞和GC B细胞的比例亦有增加。ELISA的结果显示，从小鼠脾脏的GC B细胞中筛选到具有高亲和力的嵌合抗体。该抗体轻链的第76位苏氨酸替换成了异亮氨酸，且第98位苯丙氨酸发生缺失。随后，ELISA结果显示，该嵌合抗体具有较强的亲和力，轻链氨基酸没有变化的抗体和轻链仅携带第98位氨基酸缺失的抗体的亲和力较弱，而轻链仅携带第76位氨基酸替换的抗体的亲和力介于嵌合抗体和轻链氨基酸没有变化的抗体之间。假病毒中和实验的结果显示，该嵌合抗体的半数抑制浓度（half maximal inhibitory concentration，IC₅₀）为0.995 ng/μL；轻链仅携带第76位氨基酸替换的抗体的IC₅₀为1.724 ng/μL，中和能力有所减弱；轻链仅携带第98位氨基酸缺失的抗体的IC₅₀为71.05 ng/μL，轻链氨基酸没有变化的抗体的IC₅₀为42.06 ng/μL，中和能力均较弱。结论·成功筛选得到对SARS-CoV-2具有中和能力的人鼠嵌合抗体，且该抗体轻链上第76位苏氨酸替换为异亮氨酸是决定其中和能力的关键。

Objective ·To screen the human-mouse chimeric antibodies that neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and analyse the key amino acid site changes in their light chain. Methods ·Antibody heavy chain gene-humanized mice were immunized with the spike protein of SARS-CoV-2. Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay were performed to analyse the antibody titer in serum level. Flow cytometry was used to sort plasma cells and germinal center (GC) B cells from the mouse spleen and lymph nodes, and the proportions of different B cells were analysed. RNA was extracted to construct a mouse-origin antibody light chain gene library, which was paired with the knocked-in human-origin antibody heavy chain gene for antibody expression. ELISA was employed to screen for high-affinity chimeric antibodies. Amino acid changes in the light chain of high-affinity chimeric antibodies were analysed by comparing them with mouse-origin germline antibody light chain genes. Chimeric antibodies with single amino acid changes in the light chain were constructed and expressed, and their affinity and neutralization abilities were tested through ELISA and pseudovirus neutralization experiments. By comparing the affinity and neutralization abilities of the chimeric antibodies with or without single amino acid changes in the light chain, the key single amino acid change was analysed. Results ·Compared to unimmunized mice, the immunized mice showed an increased titer of specific antibodies against SARS-CoV-2 on the serum level, along with an elevated proportion of plasma cells and GC B cells in the spleen and lymph nodes. ELISA showed that a high-affinity chimeric antibody was screened from GC B cells in the mouse spleen. The light chain of the antibody had a 76^th serine-to-isoleucine substitution and a 98^th phenylalanine deletion. Another ELISA showed that the chimeric antibody exhibited high affinity, the antibody without amino acid change on the light chain and the antibody with only the 98^th phenylalanine deletion showed low affinity, and the antibody with only the 76^th serine-to-isoleucine substitution demonstrated intermediate affinity between the chimeric antibody and the antibody without amino acid change on the light chain. Pseudovirus neutralization experiments revealed that the chimeric antibody had a half-maximal inhibitory concentration (IC₅₀) of 0.995 ng/μL; the antibody with only the 76^th serine-to-isoleucine substitution had an IC₅₀ of 1.724 ng/μL, indicating a slight decrease in neutralization ability; the antibody with only the 98^th phenylalanine deletion had an IC₅₀ of 71.05 ng/μL; the antibody without amino acid change on the light chain had an IC₅₀ of 42.06 ng/μL, suggesting weak neutralization ability. Conclusion ·For the screened human-mouse chimeric antibodies that neutralize SARS-CoV-2 in this study, the key amino acid change determining the neutralization ability of the chimeric antibody is the 76^th serine-to-isoleucine substitution.