摘要
目的：构建抗人精浆蛋白的单链抗体基因，可为进一步构建、表达抗人精浆蛋白单链抗体／羧肽酶Ａ融合蛋白，用于前列腺癌的抗体导向酶－前体药物疗法奠定基础。方法：利用加端ＰＣＲ技术，在已克隆的抗人精浆蛋白单克隆抗体ＶＨ和Ｖκ基因两端加上限制性酶切位点，再分别克隆入载体ｐＵＣ１９－ｌｉｎｋｅｒ中，构建ＶＨ－ｌｉｎｋｅｒ－Ｖκ形式的Ｅ４Ｂ７单链抗体基因。利用全自动荧光测序仪测定其序列，采用ＰＣ／Ｇｅｎｅ软件与已知的ＶＨ和Ｖκ基因进行核苷酸序列的同源性比较，并推导其编码的氨基酸序列。结果：Ｅ４Ｂ７单链抗体基因全长为７４１ｂｐ，为一开放读框，编码２４７个氨基酸，与已知的抗人精浆蛋白单克隆抗体ＶＨ和Ｖκ基因完全同源。ＶＨ和Ｖκ间有４５ｂｐ的ｌｉｎｋｅｒ序列，推导的氨基酸序列为（Ｇｌｙ４Ｓｅｒ）３，与设计的序列相符。结论：构建成功序列正确的抗人精浆蛋白单链抗体基因，为构建。
Aim: To construct the single chain variable fragment (ScFv) gene specific for human γseminoprotein, for the genetic construction and expression of a fusion protein of the ScFv and carboxypeptidase A, which could be used in antibodydirected enzyme prodrug therapy for prostatic cancer. Methods: The restriction enzyme sites were added on the previously cloned VH and Vκ genes of antihuman γseminoprotein monoclonal antibody by addon PCR. The VH and Vκ genes were sequentially cloned into the vector pUC19linker to construct the E4B7 ScFv gene in a form of VH linkerVκ. The E4B7 ScFv gene was sequenced with autoDNA sequencer and analysed with PC/Gene. Results: The E4B7 ScFv gene consisted of 741 bp, encoding 247 amino acids.A 45 bp linker sequence was found between VH and Vκ and the predicted amino acid sequence of the linker was (Gly4Ser)3. Conclusion: The correct gene of antiγseminoprotein ScFv was constructed.It would be used to construct the antiγseminoprotein/ carboxypeptidase A bifunctional antibody.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
1998年第3期191-194,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
全军重点实验室基金
