| Immunotoxin is coupling of a monoclone antibody which can high specially binding with the receptor or antigen on the surface of tumor cell and a molecule that is toxic to the tumor cell.Cobra venom cytotoxin(CTX),which is purified from Naja naja atra venom,are the major active protein found in cobra venoms and constitute up to about 40% of total protein in crude venom.They are composed of 60-63 amino acid resides with mol.wt 7000Da and has been the topic several domestic and foreign reports,confirming its strong anti-tumor effects.The observation that the IL-4 receptor(IL-4R) is highly expressed on the surface of pancreatic cancer cells,but not in normal pancreatic tissue[1] provides an opportunity for mAb-base targeting of these tumors.Current efforts utilizing an immunotoxin targeting IL-4R have achieved a series of encouraging results[2, 3]. To explore the toxic effect of CTX on pancreatic cancer cells and observe whether it is possible to selectively kill pancreatic cancer cells by virtue of their high-level expression of IL-4R, we coupled CTX to an anti-IL4R mAb (MAIL4R) and compared the selective killing effects on PANC-1 human pancreatic ductal carcinoma, BxPC-3 human pancreatic cancer cells, and human lung adenocarcinoma H1299 cells (which do not express IL-4R) in the hopes of providing experimental data for a new treatment method against pancreatic cancer. Isolation and Purification of CTX In this study,Naja naja atra venom was isolated on SP Sepharose FF cation-exchange colum fractionated into 13 protein peaks.The eleventh frations could induce the rat isolated Langendraff's preparations to decrease their contracture amplitudes, ultimately leading to complete cardiac arrest.They acted on the rat isolated phrenic nerve-diaphragm preparations and indicated no Nerve-Muscle block but could inhibit direct contracture of diaphragm.Fraction XI was futher purified by Sephasil Peptide C18.It migrated as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) by coomassie brilliant blue stains which showed that it was homogeneous with one band.The mol.wt was about 7235Da,and it exhibited on detectable phospholipaseA2(PLA2) activity with the PH-stat technique.The results showed that showed that the fraction XI was CTX and then we named him CTX-XI. The construction of immunotoxin between CTX and MAIL4R CTX and MAIL4R were couple using the N-succinimidyl amide-3-(2-pyridyl disulfide) propionate (SPDP) method. Both the antibody and cytotoxin activity were confirmed to be maintained in the conjugate. MAIL4R-CTX, was producing a single band by SDS-PAGE with molecular weight greater than 200 kD; after reduction, 2 bands were observed. The conjugate had strong reactivity with anti-cobra venom serum, as well as with an anti-mouse IgG antibody. Immunocytochemistry using this conjugate stained BXPC-3 human pancreatic cancer cells, while H1299 human lung cancer cells, which do not express IL-4R, were not stained. Assay of cytotoxicity in vitro.MTT assay was used to observe the in vitro cytotoxic activity of CTX,MAIL4R and immunotoxin towards PANC-1,BXPC-3 and H1299 cells. The CTX moiety displayed rapid cytotoxic effects on PANC-1,,BXPC-3 and H1299 cells. For instance, at a concentration of 8μg/ml, the rate of cell death for PANC-1,BXPC-3, and H1299 cells was 89.8%,88.0%, and 89.3%, respectively, with no obvious difference between the 3 lines. Monoclonal antibody against the interleukin-4 receptor had no intrinsic toxic effect on these cells. At a concentration of 9.4μg/ml, the cytotoxicity rate of CTX-MAIL4R on PANC-1 and BXPC-3 cells was, 73.1% and 84.4%, respectively, while it was only 29.8% for H1299 cells.Conclusion: One purified CTX isolated from Naja naja venom containing no PLA2 activity was obtained by SP Sepharose FF cation-exchange chromatography and Sephasil Peptide C18.The MAIL4R-CTX immunotoxin was successfully constructed by SPDP conjugation and had selective cytotoxicity against pancreatic cancer cells that express high levels of IL-4R.
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