摘要
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-linker-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 syndrome virus（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 overlapping-PCR.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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第6期461-464,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家行业科技计划项目(200903055)
国家自然科学基金(30871857
U0931003/L01)
山东省自主创新成果转化重大专项(2008ZHZX1A1101)
关键词
猪繁殖与呼吸综合征
抗独特型抗体
单链抗体
porcine reproductive and respiratory syndrome virus
anti-idiotype antibody
scFv
