嵌合内含子对抗bFGF抗体基因在293T细胞中表达的影响

目的：构建含嵌合内含子和抗bFGF抗体基因的真核表达载体并在293T细胞中表达，探讨嵌合内含子对抗体表达的影响。方法：设计引物分别从载体pCl-neo和pComb3-Fab25中扩增出内含子和抗体Fd段、κ链基因序列，按不同组合构建到含人免疫球蛋白IgG1恒定区Fc段基因的表达载体pIgG中。重组质粒经脂质体PEI转染293T细胞，荧光显微镜观察质粒转染情况，采用夹心ELISA和Western blot检测细胞上清中抗体的表达。结果：DNA测序和酶切分析表明，内含子和抗体基因成功插入pIgG，获得了重组质粒pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron和pIgG-Fd-intron-κ-intron。倒置荧光显微镜下可观察EGFP大量表达，Western blot分析上清中有抗体的表达，夹心ELISA检测pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron和pIgG-Fd-intron-κ-intron抗体表达量分别为1.21mg/L、0.468mg/L、7.39mg/L、0.601mg/L。结论：成功构建了含嵌合内含子和抗体基因的真核表达载体并在293T细胞中得到表达。嵌合内含子插入κ链基因5’端可提高抗体的表达，插入Fd段5’端则抑制抗体的表达。

Effect of Chimeric Intron on the Expression of Anti-bFGF Antibody Genes in 293T Cells

Objective: To construct eukaryotic expression vectors containing genes of chimeric intron and anti-bFGF antibody and express in 293T cells for exploring the effect of chimeric intron on antibody expression. Methods: Genes of chimeric intron and Fd、κ chain were amplified from vectors pCl-neo and pComb3-Fab25 respectively, then inserted in different combinations into expression vector pIgG containing human immunoglobulin IgG_1 Fc region. Recombinant plasmids were transfected into 293T cells by liposome polyethyleneimine (PEI). Transfection result was observed by fluorescence microscope and antibody expression was analyzed by sandwich ELISA and Western blot. Results: The results of sequencing and restriction enzyme digestion analysis showed that intron and antibody genes were successfully inserted into expression vector plgG. Fluorescence observations indicated that plasmids were transfected into 293T cells and antibody in culture supernatant was detected by Western blot. Antibody concentration of plgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron and plgG-Fd-intron-K-intron was 1.21mg/L、0.468mg/L、7.39mg/L、0.601mg/L respectively. Conclusion; Recombinant expression vectors containing chimeric intron and antibody genes were constructed and expressed in 293T cells successfully and the chimeric intron may conduct important action. Antibody expression can be improved by inserting chimeric intron into 5' upstream of k chain, but depressed into 5' upstream of Fd.