抗人红细胞H抗原单链抗体基因克隆和表达

Cloning and expression of the variable region genes of the monoclonal antibody against H antigen on human erythrocyte

目的克隆抗人红细胞H抗原单克隆抗体轻、重链可变区(VH、VL)基因并构建单链抗体(ScFv)基因及其表达载体,实现其在原核细胞中的表达。方法从分泌抗人红细胞H抗原单克隆抗体的杂交瘤细胞株2E8中提取总RNA,采用RT-PCR法获得抗人红细胞H抗原单克隆抗体的VH、VL基因;利用重叠引物延伸法(splicing by overlap extension,SOE)将轻重链可变区基因连接,并引入连接肽(Linker)编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因,将其克隆到原核表达载体pET-his中,转化BL21(DE3)plysS细胞,IPTG诱导表达;获得的目的蛋白经纯化后,用SDS-PAGE与Western blotting对目的蛋白进行鉴定,间接ELISA、竞争ELISA和免疫荧光法检测目的蛋白的活性。结果克隆的VH基因长度为351bp,属于鼠抗体可变区重链基因家族I(B)亚群;VL基因长度为339bp,属于鼠抗体可变区轻链基因家族I亚群;SOE法克隆的单链抗体基因为750bp;构建的含原核表达载体的菌株经IPTG诱导后,SDS-PAGE及Western blotting法检测到Mr32000的目的蛋白(表达的ScFv);ScFv纯化后,经免疫荧光法、间接与竞争ELISA法检测到该ScFv蛋白具有生物结合活性。结论成功地克隆了抗人红细胞H抗原单克隆抗体VH与VL基因和ScFv基因,构建ScFv基因的表达载体,实现了ScFv在大肠杆菌BL21(DE3)plysS细胞中的活性表达,为基于红细胞H抗原的免疫检测技术建立奠定了基础。

Objective To clone the variable region genes of the monoclonal antibody against H antigen on human erythrocyte for assembling single chain Fv （ ScFv ） gene and expressed in E. coli cells. Methods RT-PCR was used to clone the immunoglobulin（Ig） genes encoding variable regions of heavy and light chains （ VH and VL ） from the hybridoma cell line 2E8, which secrets the monoclonal antibody against H antigen on human erythrocyte. The two variable region genes were spliced by overlap extension and assembling ScFv gene encoding the anti-H antigen. The ScFv was cloned into expression vector pET-his and expressed in BL21 （DE3） plysS cells. The target protein was identified by SDS-PAGE and Western blotting. Immunofluorescence, indirect ELISA, and competitive ELISA were performed for detecting the binding activity of the target purified protein. Results The VH gene was consisted of 351 bp and the deduced amino acid sequence was closely related to the published sequences of the variable region Ⅰ（B） subgroup of mouse Ig heavy chain. The VL gene was consisted of 339 bp and the deduced amino acid sequence was very similar to the published sequences of the variable region Ⅰ subgroup of mouse Ig kappa light chain. The assembled ScFv gene was consisted of 750 bp including the sequence of linker peptide. A new Mr 32 000 protein band was found by SDS-PAGE and Western blotting after expression in BL21 （DE3） plysS cells. The binding activity of SeFv was identified by immunofluoreseence and competitive ELISA. Conclusion The variable genes are cloned successfully. The ScFv gene is successfully assembled and expressed in BL21 （DE3） plysS cells, which would make substantial contribution to the potential development of antibody-directed immanoassay based on H antigen on human erythrocyte.