重组鸡Igλ轻链真核细胞分泌表达及其单克隆抗体的制备与鉴定

目的: 构建重组载体pcDNA-λ并在COS7细胞分泌表达鸡Igλ轻链, 制备抗鸡Igλ轻链的单克隆抗体(mAb).方法: PCR扩增带有信号肽的鸡Igλ轻链基因, 限制性酶切消化后, 定向克隆于真核表达载体pcDNA3, 构建具有6×His标签、分泌表达的重组载体pcDNA-λ.重组载体转染COS7细胞后, SDS-PAGE分析真核表达载体pcDNA-λ在COS7细胞的分泌表达.用2×106个雏鸡B淋巴细胞免疫BALB/c小鼠, 取脾细胞与骨髓瘤细胞融合, 分别采用细胞免疫荧光和间接ELISA筛选阳性杂交瘤克隆.结果: 成功构建分泌表达鸡Igλ轻链的重组载体, SDS-PAGE分析表明在COS7细胞上清有目的蛋白的分泌表达.杂交瘤细胞经筛选与鉴定, 获得2株分泌抗鸡Igλ轻链mAb的杂交瘤细胞株, Western blot检测到该mAb对重组鸡Igλ轻链的反应性.结论: 构建了重组表达载体pcDNA-λ并在COS7细胞上清分泌表达了重组鸡Igλ轻链.并用淋巴细胞杂交瘤技术成功筛选出抗鸡Igλ轻链mAb.为深入研究鸡Igλ轻链的结构与功能奠定了基础.

Secretable eukaryotic expression of recombinant chicken Igλ and preparation of monoclonal antibodies against chicken Igλ

AIM: To construct the eukaryotic expression vector for chicken Iglambda light chain, to express it on COS7 cells and to prepare the monoclonal antibodies against chicken Iglambda. METHODS: The cDNA of chicken Iglambda light chain with signal peptide sequence was amplified and then inserted into eukaryotic expression plasmid pcDNA3 after double enzyme cutting. The constructed recombinant vector was transfected into COS7 cells by lipofectamin and the secretable eukaryotic expression of chicken Iglambda light chain was verified by Western blot. The monoclonal antibodies (mAbs) against chicken Iglambda light chain were prepared by immunizing BALB/c mice with 2x10(6) chicken B cells and by cell fusion technology. RESULTS: The eukaryotic expression vector was successfully constructed. Western blot demonstrated that chicken Iglambda light chain existed in the cultural supernatant. The hybridoma lines secreting anti-Iglambda mAbs were screened by indirect ELISA. The specific reactivity between anti-Iglambda mAbs and recombinant chicken Iglambda light chain was detected by Western blot. CONCLUSION: The secreted recombinant chicken Iglambda light chain and anti-Iglambda mAbs provide a basis for further study of the functions of chicken Iglambda.