抗CEA和CA19-9的单链抗体在HEK293细胞中的融合表达及亲和力鉴定
DOI: 10.3971/j.issn.1000-8578.2019.18.0824
Keywords: One Case Report,Evaluation Value of Tissue Polypeptide Antigen Combined with ProGRP, CEA, NSE,SCC and CYFRA21-1 on Patients with Lung Cancer,Specific Biomarkers and Related Gene of Medullary Thyroid Carcinoma,Serum VEGF-C Combined with CA125 Assay for Detecting Lymph Node Metastasis of Ovarian Cancer,CA19-9, CEA and CA125 as A Predictor for Response and Survival in Unresectable Pancreatic Cancer Patients Treated with Chemoradiotherapy,Expression and Clinical Significance of CEA mRNA in Peripheral Blood of Non-small Cell Lung Cancer Patients,Construction of Gene Library of Human-Originated Single-Chain Variable Fragment Antibody Associated with Esophageal Cancer,Expression, purification and functional detection of anti-HCC hdsFv fused with RC-RNase,Diagnostic and Therapeutic Value of Serum CA125,CA19-9 and CEA in Colorectal Carcinoma,Construction and Preliminary Identification of Single-chain Fv Antibody against Human Hepatocellular Carcinoma,Antitumor Effect of Cytosine Deaminase Genetherapy in Situ Human Colon Carcinoma in Nude Mice,Clinical Applicated Investigation of Tumor Marker CA72-4,CA19-9 in Digestive Tract (Cancer),Induction of CEA-Specific cytotoxic T lymphocytes by dendritic cells transfected with CEA-recombinant vaccinia virus
Abstract:
摘要 目的 在HEK293细胞中高效表达抗CEA scFv和抗CA19-9 scFv两种单链抗体的Fc融合蛋白（anti-CEA scFv-Fc和anti-CA19-9 scFv-Fc），并测定两种单链抗体亲和力。方法 从GenBank中获取两种scFv基因序列；经优化后合成，并与pET32a（+）构成原核表达载体anti-CEA scFv/pET32a（+）和anti-CA19-9 scFv/pET32a（+），在E.coli BL21（DE3）中进行IPTG诱导表达；两种scFv经PCR扩增技术删除末端终止序列，利用重叠区基因扩增法在anti-CEA scFv上游加入人IL-2信号肽，分别与含人IgG Fc段基因的pTT5载体（Fc/pTT5）构成真核表达载体（anti-CEA scFv（-t）-Fc/pTT5、IL2-anti-CEA scFv（-t）-Fc/pTT5和anti-CA19-9 scFv（-t）-Fc/pTT5）；使用脂质体转染法，将3个重组质粒转入HEK293细胞中进行表达；产物经rProtein-A FF亲和层析纯化，竞争性细胞ELISA和Western blot检测。结果 免疫印迹显示，3个scFv-Fc融合蛋白均在HEK293细胞中表达；竞争结合实验表明，anti-CEA scFv-Fc和anti-CA19-9 scFv-Fc的抗体浓度分别为8.2 nmol/L和6.0 nmol/L。结论 anti-CEA scFv和anti-CA19-9 scFv能在HEK293细胞内表达；虽然表达量较低，但纯化后表达产物能够识别肿瘤细胞表面抗原CEA和CA19-9
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