CAJ | 학술논문

目的克隆抗人核内不均一核糖核蛋白A2/B1(heterogeneous nuclear ribonucleoprotein A2/B1,HnRNPA2/B1)单克隆抗体的重链可变区(variable region of heavy chain, VH)和轻链可变区(variable region of light chain, VL)基因,构建抗HnRNPA2/B1单链抗体(single chain Fv,ScFv)基因,并在大肠杆菌表达.方法采用斑点ELISA、Western印迹和免疫组化检测抗HnRNPA2/B1单克隆抗体3E8的特异性,并通过逆转录-聚合酶链反应、重叠延伸PCR来克隆、串联VH和VL基因,构建抗HnRNPA2/B1 ScFv基因pET28(a+)表达载体,在大肠杆菌BL21中诱导表达,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、竞争抑制ELISA检测表达产物.结果 3E8抗体能特异性地与HnRNPA2/B1合成肽以及3株人肺癌细胞内HnRNPA2/B1结合;克隆的VH基因为345 bp,VL基因为309 bp,符合小鼠抗体可变区特性;抗HnRNPA2/B1 ScFv蛋白以包涵体形式表达,分子量约28 000,并具有与抗原结合活性.结论成功构建了抗HnRNPA2/B1 ScFv基因克隆,并在大肠杆菌中获得了功能性表达,为阐明HnRNPA2/B1与肺癌相关性奠定了实验基础.
목적극륭항인핵내불균일핵당핵단백A2/B1(heterogeneous nuclear ribonucleoprotein A2/B1,HnRNPA2/B1)단극륭항체적중련가변구(variable region of heavy chain, VH)화경련가변구(variable region of light chain, VL)기인,구건항HnRNPA2/B1단련항체(single chain Fv,ScFv)기인,병재대장간균표체.방법채용반점ELISA、Western인적화면역조화검측항HnRNPA2/B1단극륭항체3E8적특이성,병통과역전록-취합매련반응、중첩연신PCR래극륭、천련VH화VL기인,구건항HnRNPA2/B1 ScFv기인pET28(a+)표체재체,재대장간균BL21중유도표체,통과십이완기류산납취병희선알응효전영、경쟁억제ELISA검측표체산물.결과 3E8항체능특이성지여HnRNPA2/B1합성태이급3주인폐암세포내HnRNPA2/B1결합;극륭적VH기인위345 bp,VL기인위309 bp,부합소서항체가변구특성;항HnRNPA2/B1 ScFv단백이포함체형식표체,분자량약28 000,병구유여항원결합활성.결론성공구건료항HnRNPA2/B1 ScFv기인극륭,병재대장간균중획득료공능성표체,위천명HnRNPA2/B1여폐암상관성전정료실험기출.