The Research Of Preparation Of Chimeric Antibody Against Tissue Factor

Tissue factor (TF), a transmembrane glycoprotein with a molecular mass of 47 kD, plays an important role of coagulation within vessel wall。Recent research demonstrated that TF contributes to the pathogenesis of atherosclerosis,angiogenesis and tumor growth。Monoclonal antibody anginst TF can specifically inhibit activity of TF, thus inhibiting tumor growth。Since murine antibody has significant immunogenicity when used as drugs on humans, its widespread clinical application is greatly limited。Use of genetic engineering techniques to produce recombinant chimeric or humanized antibodied is a key approach to therapeutic antibodies。Therefore, in this paper, based on murine anti-TF monoclonal antibody established by our laboratory, we constructed and expressed a human-mouse chimeric antibody against human TF in CHO-K1 cells, and achieved the expression of TF chimeric antibody in a stable CHO cell lines (JJ3)。This recombinamt antitibody was purified by Protein A affinity chromatography and in vitro analysis indicated that it had anticoagulant activity and significantly inhibited the growth of tumor cell lines K562 and HL-60。This chimeric antibody could have a potential clinic application。Fist of all, total RNA was extracted from the murine hybridoma cell line secreting anti-TF monoclonal antibody。The VH and VL genes were amplified by RT-PCR。Then the genes of chimeric VH and VL were subcloned into plasmid pCN40 to construct a co-expressing recombinant plasmid pCN40-VH-VL, containing signal peptide sequences and constant regions of human immunoglobulin IgG1。The recombinant plasmids were digested by AgeⅠ, BstBⅠand HpaⅠ, EcoRⅠand agarose gel electrophoresis showed that the VH and VL genes fragments are approximately 300 bp in accordance with the theoretical value (321bp,351bp)。VH and VL genes were sequenced and analyzed by automatic DNA sequencer to comfirm their sequences。This indicates that we have successfully constructed mouse-human chimeric antibody plasmid pCN40-VL-VH against human TF。Secondly, restructuring expression plasmid of pCN40-VH-VL was extrated by Endo-free Plasmd Midi Kit and transfected into CHO-K1 cells by using LipofectaminTM 2000 Reagent, and cells were cultured in the presence of G418(800μg/mL) for two weeks。The cultural supernatant was collected and detected by ELISA and Western blot。Then the cells were screened by subcloningo CHO-ch-TF cells were cultured large scale by DMEM medium。The cultural supernatant was collected and purified by Protein A affinity column。A CHO cell line that constantly secreted chimeric antibody against TF was obtained。RT-PCR showed that recombinant genes had been cloned into CHO cells exactly。ELISA and Western blot showed that human-mouse chimeric antibody against TF maintained the binding activity and specificity to human TF molecule。The concentration of anti-TF human-mouse chimeric antibody in cell supernatants was about 50 mg/L The molecular weight of heavy chain and light chain of anti-TF chimeric antibody is about 50 kD and 26 kD and purity is more than 97% as evaluated by SDS-PAGE。Finally, to examine the inhibitory effect on tumor cell growth by chimeric TF antibody, we chose the K562. HL-60,LOVO and BEL-7402 cells as targets。These cell lines were cultured in the presence of human-mouse chimeric antibody against TF at 72 h and cell growth was monitored by MTT assay。The results showed that anti-TF chimeric antibody could inhibit proliferation of K562 and HL-60 cells in vitro o The maximum inhibitory rate was 35% and 64.9%。But it did not affect the proliferation of LOVO and BEL-7402 cells。Different concentrations of TF chimeric antibody were respectively incubated in the 37℃water with same concentration of TF at 1.5 h。The results of PT detection showed that clotting time of healthy human plasma was longer significantly as the concentration of TF chimeric antibody increasing。In conclusion, we constructed and expressed a human-mouse chimeric antibody against human TF in CHO-K1 cells successfully。The cultural supernatant purified by Protein A affinity column and the antibody is more than 97% pure。In vitro analysis indicated the chimeric antibody has anticoagulant activity and significantly inhibits the growth of K562 and HL-60 cells。...