VEGF的单抗制备及胃癌细胞MGC803表达VEGF的鉴定

Preparation of monoclonal antibodies to human vascular endothelial growth factor(VEGF 121 ) and identification of its expression on gastric carcinoma cell line MGC803

目的从胃癌细胞ＭＧＣ８０３克隆并在体外表达血管内皮细胞生长因子（ＶＥＧＦ），利用ＶＥＧＦ１２１单克隆抗体对其产物进行鉴定，为进一步明确肿瘤组织中ＶＥＧＦ来源，并以ＶＥＧＦ为靶点治疗肿瘤奠定基础。方法用杂交瘤技术制备ＶＥＧＦ１２１单克隆抗体（ＭｃＡｂ），通过ＥＬＩＳＡ、３ＨＴｄＲ掺入检测其活性；用ＲＴＰＣＲ从ＭＧＣ８０３细胞克隆ＶＥＧＦ基因，与原核表达载体ＰＧＥＸ２Ｔ重组，在大肠杆菌中进行表达，以Ｗｅｓｔｅｒｎｂｌｏｔ对表达产物进行鉴定。结果所制备的ＶＥＧＦＭｃＡｂ能特异性地同ＶＥＧＦ结合，并能中和ＶＥＧＦ对内皮细胞的促增殖作用；利用ＲＴＰＣＲ扩增出特异的ＶＥＧＦ基因片段，重组到ＰＧＥＸ２Ｔ表达载体中，在大肠杆菌ＸＬ１ｂｌｕｅ中得到了稳定高效表达的蛋白产物，并能特异性地被ＶＥＧＦ抗体所识别。结论在ＭＧＣ８０３细胞中存在ＶＥＧＦｍＲＮＡ表达，提示肿瘤细胞是肿瘤组织中ＶＥＧＦ的重要来源。ＶＥＧＦＭｃＡｂ不仅可对其存在进行检测，而且可有效抑制ＶＥＧＦ对内皮细胞的促增殖作用。

Objective To further elucidate the source of VEGF in solid tumors. Methods Traditional hybridoma technology was used to prepare VEGF monoclonal antibodies (McAb); It's activity was measured by 3 H TdR incorporation on human umbilical vein endothelial cells (HUVEC). VEGF gene was amplified by PCR with cDNA as template from MGC803 cell lines. The PCR products were cloned into fusion protein prokaryotic expression vector PGEX2T which expressed in E.coli XL 1blue. Western blot analysis was used to identify expression of VEGF. Results The McAbs could specifically bind to VEGF in ELISA. One of the McAbs(5C 5) could neutralize the mitogenic activity of VEGF 121 on HUVEC in a dose dependent manner. The VEGF gene fragment obtained from RT PCR cloned to PGEX2T vector expressed a protein product which reacted specifically with McAb 5C 5. Conclusion Gastric carcinoma is shown to express VEGF. It may be a major source of VEGF in human solid tumor. Monoclonal antibody against VEGF may be of value in the treatment of cancer.