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目的:构建人源肝癌单链抗体(scFv)基因真核表达载体pSectag2/scFv并在中国仓鼠卵巢(CHO)细胞中获得表达。方法:利用噬菌体细胞内重组技术筛选得到了肝癌特异性scFv,用PCR技术及核酸内切酶技术将其克隆入真核表达载体pSectag2,构建重组载体pSectag2/scFv,测序鉴定后,电转染CHO细胞,利用SDS-PAGE和Western blot检测目的蛋白的表达情况。结果:将750bp人源肝癌scFv插入真核表达载体pSectag2,经DNA测序鉴定正确,成功构建了pSectag2/scFv。将pSectag2/scFv转染CHO细胞后获得目的蛋白表达。SDS-PAGE和Western blot检测表明目的蛋白Mr约为34000。结论:成功地构建抗scFv真核表达载体pSectag2/scFv,并在CHO细胞中获得表达,为人源肝癌scFv的进一步的应用研究提供依据。
OBJECTIVE: To construct the eukaryotic expression vector pSectag2 / scFv of human hepatocellular carcinoma single chain antibody (scFv) gene and to obtain its expression in Chinese hamster ovary (CHO) cells. METHODS: HepG2-specific scFv was screened by phage intracellular recombination technique. The recombinant plasmid pSectag2 / scFv was cloned into the eukaryotic expression vector pSectag2 by using PCR and endonuclease techniques. After sequencing, it was electroporated into CHO Cells, the use of SDS-PAGE and Western blot detection of the expression of the target protein. Results: The 750bp human hepatocellular carcinoma scFv was inserted into the eukaryotic expression vector pSectag2, which was identified by DNA sequencing. The pSectag2 / scFv was successfully constructed. The target protein was obtained after transfection of pSectag2 / scFv into CHO cells. SDS-PAGE and Western blot showed that the target protein Mr was about 34000. CONCLUSION: The eukaryotic expression vector pSectag2 / scFv against scFv was successfully constructed and expressed in CHO cells, which provided a basis for further application of scFv in human hepatocellular carcinoma.