Anti-ni-35 Recombinant Single-chain Antibody Expression Vector And Preliminary Expression

Axons in the central nervous system (CNS) of higher vertebrates generally fail to regenerate after injury. This lack of regeneration is crucially influenced by neurite growth inhibitory protein constituents of CNS myelin. The myelin-associated protein NI-35 exerts a powerful inhibition on axon regrowth. We have been shown previously that a monoclonal antibody (anti-NI-35 mAb) capable of binding and neutralizing NI-35, can induce the long-distance axonal regeneration (more than 7 mm at 3 weeks after injury) and increased structural plasticity. But the monoclonal antibody has many shortcomings in clinical or lab application; big size and large molecular weight; poor blood brain barrier penetration capability; serious immunological problem etc. This study was designed to recombine the genes from the variable regions of light chain and heavy chain of mAb anti-NI-35, a monoclonal antibody against rat NI-35, by a short peptide (Gly4Ser)3 to construct a new anti-NI-35scFv gene. This anti-NI-35 scFv protein was expressed in Escherichia coli BL21 as a fusion protein, equipped with a His-tag.MethodWe want to connect the VH and VL by a well documented flexible peptide bridging sequence ( GGGGS) 3 linker peptide, and to obtain anti-NI-35 scFv gene by chemical synthesis in the VH-linker-VL orientation. The designed whole gene was divided into 35 segments, and synthesized respectively, with the length ranging from 40 bp to 50 bp.All chemically synthesized segments were assembled using extension overlap splicing PCR to obtain the whole anti-NI-35scFv gene. The PCR product was obtained, purified on an agarose gel, cut with the corresponding restriction enzymes BamHI/Hindlll, and inserted into cloning plasmid pUC18 that had been cut with BamHI/Hindlll. Colonies were selected on ampicillin-containing agar plates and plasmids were isolated from several independent clones. After judged by restriction analysis and colony PCR, the scFv gene insert was sequenced from both directions using M13F and M13R primers. The whole oper-on coding for recombinant anti-NI-35scFv gene was excised from pUCIS via BamHI and Hindlll and inserted into the expression vector PET-28a( + ). E. coli strain BL21 transformed with PET-28a( + ) was exponentially grown in a glucose mineral salt medium supplemented with elements, thiamine, and kanamycin. Fermentation was carried out at a constant temperature of 37℃. At a cell density equal to relative absorbance at 600 nm of about 0.6, recombinant gene expression was induced by the addition of IPTG and cultivation was continued for 5 hours. SDS-PAGE was performed to check the expression product.ResultSequence analysis showed that anti-NI-35scFv gene consisted of 729 bp, among them, 363 bp for heavy chain gene, located upstream of scFv gene, and 321 bp for the light chain gene, located downstream. SDS-PAGE analysis showed that the relative molecular weight of fusion protein is 31 KD, which was consistent with the theoretically predicted value.ConclusionRecombinant anti-NI-35scFv gene against rat NI-35 was successfully constructed, and large scale expressed in E. coli. BL21.