抗蓖麻毒素单链抗体基因的构建及其在大肠杆菌中的表达

为构建和表达抗RT单链抗体(ScFv)蛋白,用RT-PCR方法从能分泌特异性抗RT单克隆抗体(McAb)的杂交瘤细胞中分离纯化抗体VH和VL基因。用重叠延伸PCR方法将VH和VL拼接在一起,构建抗RT-ScFv基因。将ScFv基因连接到pMAL-p2X表达载体,转化TB1表达菌。阳性克隆用IPTG诱导18h,Western blotting鉴定重组蛋白。结果表明,试验成功扩增出了ScFv基因,长度约为750bp。通过DNA序列测定和分析,构建出VL-(Gly4Ser)3-VH。其VH全长363bp,可编码121个氨基酸,VL全长324bp,可编码108个氨基酸。SDS-PAGE和Western blotting分析结果表明,抗RT-ScFv在TB1表达菌中获得高效表达,pMAL-p2X表达的ScFv加上同时融合表达的MBP标签分子质量约为75ku。本试验成功构建了pMAL-RT-ScFv表达载体,并获得了高效表达。

Construction of a Single Chain Variable Fragment (ScFv) Antibody against Ricin and Expression in E.coli

To construct and express a single chain variable(ScFv) fragment against ricin, VH and VL genes of anti-murine RT monoclonal antibody were cloned by RT-PCR from hybridoma cell secreting anti-RT McAb. ScFv gene was spliced by sequence overlap extending (SOE) PCR. ScFv gene was cloned into pMAL-p2X expression vector and transformed into TB1 E.coli. After positive clones were induced by IPTG for 18 hours，the identification of recombinant protein was detected by Western blotting. ScFv gene of VL-(Gly4Ser)3-VH was constructed successfully. The VH chain consisted of 363 bp and encoded 121 amino acids. The VL chain consisted of 324 bp and encoded 108 amino acids. SDS-PAGE and Western blotting analysis showed that the RT-ScFv gene was expressed in TB1 E.coli. The ScFv antibody expressed by pMAL-p2X fused with MBP tag protein and the relative molecular mass of fusion protein was about 75 ku. The expression vector of pMAL-RT-ScFv fusion protein was constructed successfully，and procured high performance expression.