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目的：从分子水平分析抗人红细胞血型Ａ抗原单克隆抗体５０Ａ株，进而为制备新一代基因工程抗体的血型检定试剂奠定基础。方法：从杂交瘤细胞提取总ＲＮＡ，经ＲＴ－ＰＣＲ扩增、克隆抗体的轻、重链可变区基因，用ＰＣＲ及双脱氧核苷酸链终止法分析其核苷酸序列，采用ＰＣＲ技术重叠延伸拼接法构建５０Ａ单抗单链抗体基因。结果：序列分析表明所克隆基因为抗体轻、重链可变区基因，５０ＡＶＨ隶属于小鼠Ｉｇ重链ⅡＢ亚组，是由ＶＨ－Ｄ－ＪＨ３基因重排产生；５０ＡＶＬ隶属于小鼠ＩｇＫａｐｐａ链Ⅱ亚组，Ｊｋ１基因重排。重叠延伸拼接法构建单链抗体基因简易可行。结论：所克隆基因含有正确的抗体框架结构，可作为研究基因工程血型检定抗体的目标基因。
OBJECTIVE: To analyze the monoclonal antibody against human erythrocyte A (HLA-A) monoclonal antibody 50A at the molecular level, and to lay the foundation for the preparation of a new type of genetically engineered antibody. Methods: Total RNA was extracted from hybridoma cells and amplified by RT-PCR. The light and heavy chain variable regions of the antibody were cloned and their nucleotide sequences were analyzed by PCR and dideoxynucleotide chain termination. Construction of 50A mAb Single - chain Antibody Gene by Overlap Extension. Results: Sequence analysis showed that the cloned gene was the light and heavy chain variable region gene. 50AVH belongs to mouse Ig heavy chain ⅡB subgroup and is produced by rearrangement of VH-D-JH3 gene. 50AVL belongs to mouse IgKappa chain Ⅱ subgroup, Jk1 gene rearrangement. Overlap extension splicing method to construct single-chain antibody gene is simple and feasible. Conclusion: The cloned gene contains the correct antibody frame structure, which can be used as a target gene for the study of genetically engineered blood type antibodies.