| As known, deep vein thrombosis is one worldwide illness of high morbidity and mortality and its on-time diagnosis and corresponding treatment is of great importance. Clinical diagnosis depending on symptoms and objective signs is unreliable. A doctor needs some screening investigations including D-dimer tests, plethysmographic techniques to arrive to a final diagnose. The imaging of thrombus is becoming a routine experiment.Among many kinds of investigations for the visualization of thrombus, phlebography is called gold standard for its immediate imaging of definite location of thrombus and the blood stream state. However, the dangerous shortcoming is possible hypersensitivity, bad prognosis of complications and its difficult operation. Ultrasonography is considered to be a convenient non-invasive diagnostic method, but its low sensitivity is also estimated. Computed tomography and magnetic resonance imaging can not be widely used for their costly charge and difficult operation. The existing radioated iditope labeled fibrinogen scanning is known for its low uptaking of diagnosis. Further improvements are needed to make a specific and safe method a clinical reality.Fibrin is the major protein constituent of the blood clot and is formed from fibrinogen, a glycoprotein that circulates largely inactively, in the blood stream. Labeling a specific antibody reacting with fibrin of the clot components has already been a new nuclear imaging technique to diagnose deep venous thrombosis and investigators have been evaluated several monoclonal anti-fibrin antibodies to image thrombus and got feasible results in experimental and clinical trials. However, the current generation of MAb-based radioimmunodetection that is entering the clinic is limited in vivo due to their long circulating lives and associated effectors, e. g. HAMA. Recombinant single-chain antibodies have become the paradigm for the design of these high-affinity, protein-based targeting reagents. The results, however, were uniformly disappointing because of the loss of affinity, low clot-to-blood signal (?)atios and quick clearance. Multiple research results shows that the nonconventional length of the linker joining the two variable domains dictates the formation of multimeric scFv molecules which gain more in functional affinity and thereby improved performance including targeting and selective localization in vivo.In this experiment, the linking of the VH gene to the VL gene of anti-fibrin MAb AFkβ8E5 with a five-residue linker is planned to produce active scFv dimers with bivalent binding sites. The bivalency will lead to an increase of functional affinity and a decrease of clearance ratios in vivo which can provide a novel way to produce imaging agents.In the case of hybridoma cell line AFkβ8E5 total RNA was isolated essentially and was reverse transcribed in an appropriate reaction volume according to the manufacturer's protocols. For the amplication of VH gene and VL gene from the hybridoma, universal degenerate primers were chosen. The full length PCR products of VH gene and VL gene were purified and linked to the pMD-19T vector and sequenced by Sanger dideoxy chain-termination method. Relevant sequences were clarified to be members of the murine immunoglobulin superfamily by sequence comparing.We got assembly amplified products combined by SOE-PCR in the orientation of VL-linker-VH with another two pair of primers with restriction enzyme sites and partial linker sequence. The full length PCR product of scFv was ligated into the pMD-19T vector and transformed into E. coli DH5α. after sequenced and compared as above. Later it was digested with restriction enzymes, ligated into pET28a(+) and transformed into E. coli BL21(DE3). After induced by IPTG, the primary expression product was confirmed by SDS-PAGE and Western blot. The followed optimization of conditions for expression was done to make a good preparation for the further research.
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