| Colorectal carcinoma is one of the common malignant tumors which have higher incidence, occupying the fourth place in our country and the death rate increasing continuously in recent years. Therefore, efficient diagnosis and therapeutic approaches are important for colorectal carcinoma research. Although in recent years some progress has been made in respect to application of monoclonal antibodies for the therapy and diagnosis of colorectal carcinoma, most mAbs are of murine origin, so that they usually have antigenic activity to human , and their large sizes also lead to poor diffusion and long persistence in circulation, which severely limit the efficacy of clinical utilization. Gene engineered antibody fragments like Fab and scFv which have small molecular weight (Mr,25000 ~30000Da ) can improve diffusion and circulation clearance,while they are low avidity because of their monovalent, and their small sizes lead to too quick clearance in circulation. Recently bivalent antibody fragments which are constructed by using monovalent antibody fragment like scFv and Fab display optimized effects in tumor imaging and therapy, bivalent can promise higher a-vidity than monovalent, and the medial molecular size can guarantee the diffusion and moderate clearance in circulation.ND-lmAb is a murine monoclonal antibody against tumor-associated antigen LEA which mainly expressed in human colorectal carcinoma. It was developed by Professor Song Jindan in 1983 by immunizing Balb/c mice with CCL-187 human colorectal carcinoma cell line. The histological determination of nearly one thousand pathologic samples showed that ND-1 can bind specially to well differentiated and moderately differentiated colorectal carcinoma tissues andits specificity is superior to IgG against CEA which has been commercialized in the US. Single chain Fv of ND-1 (ND-lscFv) against human colorectal carcinoma was constructed by fusing gene of variable fragment of heavy chain and light chain and expressed in E. coli. It showed that ND-lscFv displayed fast imaging and clearance, but for the other part, monovalent and so small size lead to low avidity and low accumulation in tumor tissues, so that limit its function in tumor therapy.Therefore, we constructed the bivalent single chain Fv of ND-1 (ND-lsc (Fv)2) by genetic recombination of the gene of ND-lscFv. It was constructed by a flexible short peptide of 4 gly sines and 1 serine ( Gly4Ser) covalently connected the two scFv antibody fragments. The binding sites and molecular weight have been doubled at the same time, so as to gain high avidity and to overcome shortage of too fast clearance rate of scFv. The construction of ND-lsc( Fv)2 can provide evidence for nude mice bearing experiments and clinical diagnosis and therapy of colorectal carcinoma.Materials and MethodsFrom the expression vector pET-28a( + )ND-lscFv, the gene of ND-lscFv was amplified and added a linker sequence ( encoding Gly4Ser) to the upstream of the gene of ND-lscFv and restrict sites Sail and Hindlll by primer PI and P2. The PCR product was ligated into clone vector of pMD18-T to form pMD18-TLinker-ND-1 scFv. correct clone containing pMDlS-TLinker-ND-lscFv was i-dentified by DNA sequencing. pMD18-TLinker-scFv was double digested by Sail and Hindlll, the product of Linker-scFv was recovered and linked into pET-28a ( + ) ND-1 scFv which is prepared by double digestion by Sail and Hindlll. The novel vector pET-28a( + ) ND-lsc(Fv)2 was transformed into E. coli BL21. Condition for optimal expression was adjusted. To Express ND-lsc(Fv)2 by the optimal condition. Target protein was purified by Ni-NTA resin affinity chroma-tography, and then was refolded, its molecular weight and purity were analyzed by SDS-PAGE. Immunofluorescence Assay was conduct to determinate the specificity of ND-lsc( Fv)2to CCL-187 cell line by comparing to HeLa cell line.The avidity among ND-lmAb ND-lsc ( Fv)2and ND-lscFv was compared by ELISA.ResultsUsing the expression vector pET-28a( + )ND-lscFv as template, PCR produced a about 750bp...
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