为制备只包含抗体可变区片段(Fv)的MAb2-5G2单链抗体(scFv),本研究从分泌MAb2-5G2的杂交瘤细胞中提取总RNA,反转录制备cDNA作为模板扩增重链可变区(VH)和轻链可变区(VL)基因.以VH-1inker-VL的形式通过重叠PCR制备scFv基因,构建重组表达质粒pEasy-scFv,并在大肠杆菌中获得正确表达.通过western blot和间接免疫荧光鉴定表明scFv蛋白能够被兔抗MAb2-5G2的抗体(Ab3)识别,复性后的scFv蛋白能够特异性结合Marc-145细胞.该研究结果为进一步明确MAb2-5G2结合Marc-145细胞功能区域奠定了物质基础.%Anti-idiotype plays important roles in the immune responses against porcine reproductive and respiratory syndromevirus (PRRSV). Our previous study has shown that the monoclonal anti-idiotype antibody (MAb2) 5G2 specific to anti-PRRSV GP5 antibody could recognize and bind to Marc-145 cells. To further understand the functional domain of MAb2-5G2 for binging to Marc-145 cells, the variable light (VL) and variable heavy (VH) chain genes of MAb2-5G2 were amplified from the hybridoma cells secreting MAb2-5G2 by RT-PCR. VL and VH encoding were fused with a linker to form VH-linker-VL by overlappingPCR. VH-linker-VL was then cloned into pEasy-E1 vector and expressed in E. coli BL21. The results showed that the expressed scFv protein was recognized by the rabbit anti-MAb2-5G2 antibodies (Ab3) by western blot and the refolded scFv protein specifically bound to Marc-145 cells by indirect fluorescence assay.
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