人CD34单链抗体基因的构建及序列测定

Construction and sequence analysis of anti CD34 single chain antibody gene

目的：获得抗人ＣＤ３４单链抗体基因。方法：选用一株分泌抗ＣＤ３４抗原单克隆抗体的杂交瘤细胞株，提取总ＲＮＡ，经ＲＴ－ＰＣＲ扩增出此抗体的重链可变区和轻链可变区基因，与Ｋａｂａｔ抗体库比较得到其框架区和互补决定区的氨基酸序列；重新合成两对引物去掉保留的分泌信号前导序列并加入适当的限制性内切酶酶切位点，再经ＰＣＲ得到所需要的重链可变区和轻链可变区基因，经一弹性连接肽连接构建成抗人ＣＤ３４的单链抗体（ｓＦｖ）基因。结果：ＤＮＡ序列分析结果表明，获得的单链抗体基因的轻链可变区属于鼠κ轻链第Ⅴ亚族，重链可变区属于鼠重链第Ⅰ（Ｂ）亚族。结论：构建的抗人ＣＤ３４单链抗体基因全长为８１３个碱基，编码蛋白的相对分子质量约为３００００。

Objective: Cloning of anti CD34 single chain antibody gene. Methods: A hybridoma which can secrete anti CD34 monoclonal antibody was used in the construction of single chain antibody (sFv) gene. The cells were used for preparation of total cellular RNA and the cDNAs of the variable heavy chain (V H) and light chain (V L) regions were synthesized by RT PCR. Then framework regions (FR) and complementary determining regions (CDR) of the antibody were determined by using the Kabat antibody sequence database and new PCR primers containing appropriate restriction enzyme sites for further cloning were used to reamplify each of the V L and V H fragments, with deletion of the remaining secretory leader sequences. The DNAs of light and heavy chain were joined together via a flexible linker to obtain anti CD34 sFv gene. Results: The sequence analysis demonstrated that the V L and V H belong to kappa light chain subgroup V and heavy chain subgroup Ⅰ(B) of mouse variable region respectively. Conclusion: The complete sFv construct, including linker sequence, is 813bp and the molecular weight of encoded protein is approximately 30 kDa.