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Construction and Expression of Bivalent Single-chain Antibodies with Different Linker Sequence against Human Colorectal Carcinoma
Dan-Dan Yan1,2, Jin Fang1*, Jin-Dan Song1
1Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health of China, China Medical University, Shenyang 110001, China; 2Department of Immunology, Shenyang Medical College, Shenyang 110034, China
Abstract: To engineer and express the bivalent covalent scFv of ND-1mAb against human colorectal carcinoma， the two same ND-1scFv gene were linked by different length linker with sequences encoding G4S and (G4S)3. The expression vector pET-28a(+)ND-1sc(Fv)2 was constructed to express the ND-1sc(Fv)2 fusion protein in E.coli BL21. The expressed product was purified by metal affinity chromatography using Ni-NTA resin. The purity and immunoreactivity were analyzed by SDS-PAGE， Immunofluorescence assays (IFA) and ELISA. The results demonstrated that pET-28a(+)ND-1sc(Fv)2 gene was constructed and expressed successfully in E.coli BL2 in the form of an inclusion body. The purity of ND-1sc(Fv)2 proteins with 5 amino acid linker and 15 amino acid linker were 90% and 86% respectively. IFA and ELISA revealed that both of ND-1sc(Fv)2-5 and ND-1sc(Fv)2-15 had specific binding activity to the tumor-associated antigen LEA expressed in human colorectal carcinoma cells and obviously higher than that of ND-1scFv， and compared with ND-1sc(Fv)2-5， ND-1sc(Fv)2-15 showed the activity closer to ND-1mAb. They may become potentially useful in clinical diagnosis and therapy as a carrier for human colorectal carcinoma.
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