【摘要】 目的　利用基因工程技术将鼠源性单克隆抗体WuT9改造成单链抗体。方法　从分泌抗CD71单克隆抗体的杂交瘤细胞WuT9中提取总RNA ,采用OligodT为逆转录引物 ,逆转录合成cDNA第一链 ,然后分别采用VL 和VH 框架区的PCR引物 ,扩增VH(重链可变区 )和VL(轻链可变区 )DNA片段 ,利用事先合成的存在于VL 下游引物和VH 上游引物的部分人工连接子重叠互补序列 ,将VL 和VH 的PCR回收产物进行部分重叠PCR(SOE) ,形成单链抗体基因。最后将此基因重组进表达载体pBAD gIII C中 ,经L arabinose(左旋阿拉伯糖 )诱导表达并初步鉴定。结果　构建出 70 0bp左右的单链基因 ,并获得阳性重组表达载体克隆 ,表达产物为相对分子质量 2 70 0 0左右的蛋白分子。结论　经因特网查询表明 ,单链抗体基因重链部分属于小鼠H链可变区ⅠA亚组 ;轻链属于小鼠κ轻链可变区Ⅱ亚组 ,此单链抗体基因的表达产物具有一定的特异结合活性。本研究为抗CD71单链抗体的临床应用打下了基础。更多还原

【Abstract】 Objective To prepare murine McAb WuT9 into a si ngle-chain antibody by gene engineering technique.Methods Extr act total RNA from hybridoma cell WuT9 secreting anti-CD71 McAb and synthetize the first chain of cDNA by reverse transcription using Oligod T as a primer,then amplify V H and V L DNA fragments by PCR.Prepare the amplified V H and V L genes into a single-chain gene fragment by splicing by overlapping extension(SO E). Insert the single-chain antibody gene into expression vector pBAD/gIII/C an d express under induction of L-arabinose. The expressed product was preliminari ly identified. Results A single-chain gene fragment at a length of 700 bp was con structed,and the protein with a relative molecular weight of 27 000 was expresse d. Conclusion The heavy chain of constructed single-chain gene belongs to the subgroup ⅠA of murine heavy chain variable region,and its light chain belo ngs to the subgroup Ⅱ of murine κ light chain variable region. The expressed p roduct of the single-chain antibody showed specific binding capacity. The study laid a foundation of clinical application of anti-CD71 single-chain antibody. [更多还原