抗人β-淀粉样肽特异性单链抗体的噬菌体抗体库的构建与初步鉴定

Construction and identification of specific anti-human β-amyloid peptide ScFv by phage antibody library display system

目的:运用噬菌体展示技术构建抗人β-淀粉样肽(Aβ)特异性单链抗体的抗体库并进行初步鉴定.方法:从Aβ免疫的BALB/c小鼠脾淋巴细胞中提取总RNA,分离纯化mRNA,经逆转录合成其总cDNA.以PCR方法分别扩增出抗体的重链(VH)和轻链(VL)可变区基因片段,再经重组PCR将两基因片段由编码十五肽(Gly4Ser)3的接头连在一起,克隆到噬菌粒载体pCANTAB5E中,转化至大肠杆菌TG1,经辅助噬菌体M13KO7超感染后回收全部重组噬菌体,构建噬菌体抗体库,SDS-PAGE鉴定纯度.结果:经琼脂糖凝胶电泳分析,RT-PCR方法扩增的抗体VH和VL基因片段大小约350bp和325bp,与预期VH,VL基因片段理论值相符.经重组PCR得到大小约750bp的ScFv基因片段.ScFv基因片段经双酶切后成功定向克隆到噬菌粒载体,回收后构建成库容量为1.1×108的噬菌体抗体库,经SDS-PAGE鉴定,得到Mr约为30000ku,纯度较好的ScFv抗体.结论:成功构建了库容量大的特异性噬菌体抗体库,为抗人Aβ特异性抗体的筛选、生物活性鉴定、临床实验诊断方法建立和治疗应用打下基础.

AIM： To construct and identify the specific antihuman β-amyloid peptide ScFv by phage antibody library display system. METHODS： Total RNA was extracted from spleen B cells of BALB/c mice immunized with β-amyloid peptide, and mRNA was purified from the total RNA, then the first-strand cDNAwas synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified by primary PCR and the amplified VH and VL PCR fragments were further joined into a SeFv gene with a 15-peptide linker DNA （Gly4Ser） 3 with fill-in reactions. Then the assembled ScFv gene fragments were amplified and restriction sites were added by the second PCR. The digested SeFv gene fragments were finally ligated with the phagemids pCANTAB 5E and the phagemids containing ScFv gene were transformed into competent E. coli TG1 cells. The transformed cells were then infected with M13KO7 helper phage to rescue all recombinant phagemids with ScFv gene insert, thus an immune phage ScFv library was constructed. Purity of ScFv was analyzed by SDS-PAGE. RESULTS ： Antibody VH and VL gene fragments amplified by primary PCR were about 350 bp and 325 bp after analysis by agarose gel electrophoresis and matched the expectancy of VH and VL gene fragments. The purified VH and VL PCR fragments were further joined into a ScFv gene with fill-in reactions and reached the desired 750 bp expectancy. The recombinant phagemid with SeFv gene insert was rescued, and an immune phage ScFv library with the content of 1. 1 ×10^8 was successfully constructed. After analysis by SDS-PAGE, a purified ScFv gene was got with a relative molecular weight of about 30000 u. CONCLUSION： An immune phage ScFv library is successfully constructed. The successful preparation of anti-Aβ ScFv will serve as a useful tool for studying the etiological and pathological mechanism, diagnosis and treatment of patients with Alzheimer＇s disease.