特异性抗鼠IgD单链抗体基因的构建及序列测定

Construction and sequencing of single chain Fv of a mouse IgD specific monoclonal antibody

目的：获得特异性抗鼠 Ｉｇ Ｄ ｍ Ａｂ ４１４／ Ｄ Ｆ的单链抗体基因． 方法：在获得高亲和力抗鼠 Ｉｇ Ｄ特异性ｍ Ａｂ 重、轻链可变区基因的基础上，采用酶连法将二者拼接成单链抗体基因． Ｄ Ｎ Ａ 自动测序仪测定其核苷酸序列，进行计算机酶切位点分析及氨基酸序列推导． 结果：所构建的单链抗体基因为 Ｖ Ｈ  Ｌｉｎｋｅｒ Ｖ Ｌ 结构，长７２９ ｂｐ，其中包含重链可变区基因３５７ｂｐ， 可编码 １１９ 个氨基酸， 连接肽基因 ４５ ｂｐ， 可编码（ Ｇｌｙ４ Ｓｅｒ）３ 连接肽，轻链可变区基因３２７ ｂｐ，编码１０９ 个氨基酸，含有维持抗体结构所必须的半胱氨酸残基． 结论：获得了拼接正确、完整的抗 Ｉｇ Ｄ 单链抗体基因．

AIM: To construct a single chain Fv of a mouse immunoglobulin D specific monoclonal antibody. METHODS: The heavy and light chain variable region genes, amplified from a mouse specific anti IgD hybridoma cell line 414/DF, were connected with a flexible linker (Gly 4Ser) 3 and cloned into pUC19 plasmid. An auto DNA sequencing system then sequenced the fusion gene. RESULTS: The fusion gene consisted of a 357 bp V H, a 45 bp Linker and a 327 bp V L, indicating that a 729 bp V H Linker V L was constructed correctly. The deduced amino acid sequences were capable of encoding 243 amino acids, including the linker(Gly 4Ser) 3. CONCLUSION: The fusion gene is the scFv of the mouse immunoglobulin D specific monoclonal antibody.