抗PSMA全人源单链抗体制备、鉴定及初步应用

摘要

目的 构建抗PSMA全人源单链抗体库,筛选抗PSMA人源单链抗体(ScFv)并鉴定,应用人源ScFv进行分子显像.方法 从健康人外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人轻链和重链可变区基因,重叠PCR拼接轻重链可变区基因,构建ScFv的表达载体pDF-ScFv,电转染大肠杆菌XL1 Blue,获得噬菌体单链抗体库,酶切鉴定及测序分析,用PSMA抗原进行富集筛选,阳性克隆用Western-Blot 和DNA指纹图谱鉴定.应用直接标记法将125I标记在ScFv上,通过尾静脉注入前列腺癌动物模型体内,2h后进行小动物SPECT/CT显像,观察人源ScFv对前列腺癌的定位性能.结果 成功构建抗PSMA全人源单链抗体库,库容约2.16×108,插入片段经酶切及DNA测序证实为人抗体轻链和重链可变区基因.筛选出6株ScFv阳性克隆,Western-Blot证实6株ScFv均能与PSMA抗原特异性结合,DNA指纹图谱鉴定6株ScFv可变区基因具有多样性.动物模型的分子显像:人源ScFv能与前列腺癌PSMA抗原特异性结合,并使肿瘤显像.结论 成功构建了一个全人源噬菌体单链抗体库,并筛选出了抗PSMA单链抗体,单链抗体在动物模型体内能靶向结合前列腺癌细胞,从而为前列腺癌的靶向诊断和治疗奠定了基础.%Objective To construct a single chain antibody library against human PSMA and to screen single chain antibody against human PSMA and to perform the molecular imaging.Methods Total RNA was extracted from human peripheral blood lymphocytes before we conducted gene amplification of light chain and heavy chain variable region by RT-PCR method.The V gene were spliced to form ScFv by overlap PCR and cloned into vector pDF.The expression vectors of pDF-ScFv were constructed, and transformed into E.Coli XL1-blue to generate human phage antibody library.Enzyme digestion and sequencing analysis were performed to identify phage antibody library.PSMA antigen was used for enrichment and screening of ScFv antibodies.The positive clones were identified by Western-Blot and DNA fingerprinting.Direct labeling method was used to label 125I on single chain antibody, and then injected into the prostate cancer animal model by tail vein.After 2h, with small animal SPECT/CT imaging, the targeting ability of human ScFv antibody to prostate cancer was observed.Results We successful constructed anti PSMA scFv library with the capacity of library is about 2.16×108.The insert fragment were confirmed to be the human antibody light chain and heavy chain variable region gene by restriction enzyme digestion and DNA sequencing.We Screened out of 6 single chain antibody positive clone and western-Blot confirmed that 6 ScFv antibodies were able to bind with the PSMA antigen specifically.The DNA fingerprint identified 6 ScFv antibody variable gene regions have diversity.Molecular imaging of animal models showed: the ScFv antibody can specifically bind to PSMA antigen of prostate cancer and generate prostate tumor imaging.Conclusion A phage single chain antibody library was constructed successfully, and the anti PSMA single chain antibodies were screened out, the ScFV antibody can target prostate cancer in animal models, which laid the foundation for the targeted diagnosis and treatment of prostate cancer.