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Construction and Expression of Human Single Chain of Variable Fragment Against Hepatocellular Cancer in Eukaryotic Cell

Author: LiBingSuo
Tutor: ZuoKeFeng；ZhangFuQin；MaLongYang
School: Fourth Military Medical University
Course: Surgery
Keywords: Hepatic carcinoma Single-chain antibody CHO cells Gene Expression
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 90
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Abstract


In recent years , the gradual development of antibody library technology provides a powerful tool for the preparation of high - affinity human antibody . Single-chain antibody ( scFv ) can not know in advance, characterized in the case of the cell surface receptor and the tumor cell-specific binding , the ScFv was protein has small molecules , penetrating power , etc. . My laboratory using phage display technology to build a fully human hepatocellular carcinoma ScFv phage library , and screened to a hepatoma-specific ScFv gene in order to achieve the the ScFv gene functional expression , For further identification ScFv protein function , we take advantage of the screening the eukaryotic expression of the gene to protein . Objective: To construct the human hepatoma single chain antibody genes eukaryotic expression plasmid pSectag2B/ScFv and the further functional studies and clinical applications of hepatocellular carcinoma ScFv protein expressed in Chinese hamster ovary cells (CHO cells) , and his source to provide the basis . Methods: laboratory screening of a liver cancer -specific ScFv gene was cloned into the PCR and endonuclease eukaryotic expression plasmid pSectag2B , The recombinant plasmid pSectag2B/ScFv , sequencing , electricity transfected CHO cells , by SDS-PAGE and Western blot to detect the expression of the target protein . Results: 750 bp source hepatocellular carcinoma ScFv gene was inserted into the eukaryotic expression plasmid pSectag2B correctly identified by DNA sequencing , and successfully constructed a recombinant plasmid pSectag2B/ScFv . Protein was obtained after the pSectag2B/ScFv transfected CHO cells . SDS-PAGE and Western blot analysis showed that the target protein Mr approximately 34,000 . Conclusion : We successfully constructed with human liver cancer ScFv the recombinant plasmid pSectag2B/ScFv, and , expressed in CHO cells , and provide the basis for human source of liver cancer the ScFv the further application .

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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