抗人角蛋白全IgG基因真核表达载体的构建及表达

Construction and expression of eukaryotic expression vector for intact IgG (against) human keratin

目的 :构建抗角蛋白抗体基因的真核表达载体 ,并在CHO(dhfr-)细胞中表达。方法 :从含抗角蛋白抗体基因的原核Fab表达载体中 ,扩增VH 及VL 基因。以回收的PCR产物为模板 ,用重叠PCR扩增带有前导序列的VH、VL 基因。经XbaI/BamHI和XhoI/HindIII酶切后 ,分别插入真核表达载体pWD中 ,构建重组载体pWDκH。经PCR和测序鉴定正确后 ,用lipofectAMINE 2 0 0 0转染CHO(dhfr-)细胞。取培养上清检测抗人角蛋白全IgG的表达。结果 :构建了抗人角蛋白基因的真核表达载体pWDκH ,并在CHO(dhfr-)细胞中表达。结论 :在CHO(dhfr-)细胞中成功地表达了具有抗原结合活性的抗人角蛋白IgG 。

AIM: To construct an eukaryotic expression vector containing the gene of anti-keratin antibody and express it in CHO(dhfr^-) cells. METHODS: Both the V_L and V_H genes of an anti-keratin Fab from a phage display library were amplified by PCR.Using PCR product as the template, the V_κ and V_H genes with the leader sequences (named L_κ and L_H respectively) were amplified by overlapping PCR.After digested with endonuclease Xba I/BamH I and Xho I/Hind III, L_κ and L_H genes were inserted into pWD digested with Xba I/BamH I and Xho I/Hind III, respectively to construct pWDκH.After PCR and sequencing, the expression plasmid pWDκH was transfected into CHO (dhfr^-) cells. The culture supernatant of the transfected cells was collected and assayed for IgG activity. RESULTS: The eukaryotic expression vector pWDκH was constructed successfully and expressed in CHO(dhfr^-) cells. The expression of intact IgG against keratin was identified by ELISA and RT-PCR. CONCLUSION: The successful expression of intact IgG against keratin lays the foundation for its clinical application.