抗堆型艾美耳球虫子孢子表膜单链抗体基因的构建

Construction of ScFv gene against surface memberane antigen of Eimeria acervulina sporozoite

应用RT-PCR技术,从稳定分泌抗堆型艾美耳球虫子孢子表膜抗原的单克隆抗体的杂交瘤细胞Easp-3G3中扩增出抗体VH和VL基因,再通过重叠延伸拼接(Splice overlap extension)PCR方法在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗堆型艾美耳球虫的单链抗体基因,并将其克隆至pMD18-T载体中进行测序。经测序,基因全长为744bp,编码248个氨基酸残基;经计算机分析,VH和VL基因均为新发现的基因序列,符合功能性重排的鼠抗体可变区基因特征。结果表明,成功构建了抗堆型艾美耳球虫子孢子表面蛋白抗体的ScFv基因。

By means of RT-PCR technique,the VH and VL gene were amplified from a hybirdoma cell line Easp-3G3 producing mouse McAb against Eimeria acervulina sporozoite.The VH and VL genes were connected through a flexible hnker （Gly4Ser）3 via gene splicing by overlap extension （SOE） ,and the Vn-linker-VL（ScFv） fusion gene were cloned into a clone vector pMD18-T and sequenced.The ScFv gene were sequenced,analyzed by computer to be consist of 744 bp encoding 248 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be a new gene. The single chain variable Fv of a monoclonal antibody against Eimeria acervulina sporozoite was constructed successfully.