抗人精浆蛋白单抗κ链基因的克隆及其在HeLa细胞的表达

Cloning and expression in HeLa cells of gene encoding κ chain of an anti-human-γ seminoprotein monoclonal antibody

目的 获得鼠抗人精浆蛋白单抗（ｍＡｂ）κ链基因，并将其转染到ＨｅＬａ细胞进行表达。方法 从分泌抗人精浆蛋白ｍＡｂ的杂交瘤细胞系Ｅ４Ｂ７中提取总ＲＮＡ，用ＲＴ－ＰＣＲ法获取κ链ｃＤＮＡ。将其克隆入真核表达载体ｐｃＤＮＡ３，用脂质体转染法转染ＨｅＬａ细胞，并用免疫荧光染色法检测κ链的表达。结果从分泌抗人精浆蛋白的鼠ｍＡｂ杂交瘤细胞系Ｅ４Ｂ７中克隆了κ链基因。序列测定表明，Ｖκ属于鼠免疫球蛋白ⅩⅢ家族。以含κ链基因的真核表达载体ｐｃＤＮＡ３－Ｅ４Ｂ７κ转染ＨｅＬａ细胞后，在ＨｅＬａ细胞中检测到了κ链的表达。结论 获得了序列正确的κ链基因，并在ＨｅＬａ细胞中成功地表达，为进一步构建和表达Ｆａｂ段打下了良好基础。

Aim To obtain gene encodingκ chain of murine anti-human-γ seminoprotein monoclonal antibody and express it in HeLa cells.MethodsTotal RNA was extracted from the hybridoma cell line E4B7and subjected to reverse transcription.The amplified κ cDNA was cloned into the expression vector pcDNA3 and transfected into HeLa cells.Expressed product was detected by immunofluorescent staining. ResultsSequencing showed that the Vk gene had a high homology with mouse Ig kappa family X III.The expression vector pcDNA3-E4B7κ was expressed successfully in HeLa cells. Conclusion κ chain gene with correct sequence is obtained and expressed successfully in Hela cells. This result lays a good foundation for the construction and expression of Fab fragment of E4B7mAb.