Tumor-Targeted Gene Expression Profile And Effect Of Induction Of Tumor Cells Apoptosis Mediated By Gene Engineering Antibody Against-TfR

Part OneK562 apoptosis induced by Human-mouse Chimeric Antibody against-TfR and its MechanismObject: In the previous studies, we successfully constructed and expressed the human-mouse chimeric antibody against TfR, it also demonstrated that it has some characteristics involving in highly affinity, certain tumor-specificity localization and mediation ADCC and CDC effect. In the present study, we want to investigate that the K562 apoptosis inducted by the human-mouse chimeric antibody against TfR and its possibily mechanism.Method: â‘ The transfectomas, secreting the chimeric Ab, were resuscitated and screen by G418, then ELISA was used to test chimeric Ab expression in its culture supernatant â‘¡Antibody in ascetics was secreted by the transfectomas seeded into the abdominal cavities of Balb/c athymic mouse and purified by DEAE-Sephredax-A50 ion-exchange chromatography. â‘¢Various concentration antibodies were incubated with K562 cell and their effect on cell growth and proliferation detected by viable count and MTT. â‘£Apoptosis of K562 cell was investigated by agarose gel electrophoresis, transmission electron microscope and FCM with Annexin V-PI staining. ⑤ Antibody effect on K562 cell cycle was analysis by FCM with PI staining.â‘¥Variations of cytochrome C and activated caspase-8 in apoptosis cell's endochylema were tested by cytochrome C Release Apoptosis Assay Kit and Caspase-8 Colorimetric Assay Kits, respectively.Result: â‘ The concentration of the chimeric Ab(D2C) in the culture supernatant was about 0.5～5μg/ml in 5-day cultures when seeded at 1×10⁵ cells and the concentration of purifying D2C in ascetics was about 1～2mg/ml. It indicated that transfectomas were freezed in liquid nitrogen for two years stably secreted the human-mouse chimeric antibody against TfR (D2C). â‘¡Antibody against TfR have obviously effect on inhibition K562 cell growth and proliferation in a dose-dependent and time-dependent manner. â‘¢DNA of K562 co-incubation with Antibody against TfR display DNA ladder, their cell morphous is typical appearance of apoptosis and apoptosis rate is 13.08% ±2.01 %. All evidences demonstrated that antibody against TfR would induce apoptosis of K562 in a dose-dependent and time-dependent manner. â‘£ FCM showed that cell cycle of K562 was arrest at G1 phase by antibody against TfR. ⑤There is increase for cytochrome C but not for activated caspase-8 in endochylema of apoptosis cell, this suggested antibody against TfR induced apoptosis of K562 through the mitochondria death pathway.Part TwoConstruction, Expression and Identification of Fusionprotein TfR_scFv-GAL4Object: The purpose of the study was to construct a fusion gene TfR_scFv-GAL4 expression vector; it would further investigate expressed fusion protein TfR_scFv-GAL4 specificially binding to tumor cells expressing the TfR.Method: â‘ The TfR_ScFv gene fragment was amplified from the plasmid containing TfR_ScFv by PCR, and the GAL4 gene fragment was obtained from the plasmid pABgal4 containing GAL4, then the TfR_ScFv gene fragment and the GAL4 genefragment were cloned into the pET28a (+) expression vector and sequenced. â‘¡E.coli BL21 with recombinant plasmid TfR_ScFv-GAL4-pET expressed the TfR_ScFv-GAL4 fusion protein induced by IPTG. â‘¢The TfR_ScFv-GAL4 fusion protein was assessed by Coomassie blue staining of SDS-PAGE gels and densitometric scan, and then it was be diluteing renaturation and dislyzing renaturation. â‘£The activity of GAL4 protein fragment containing in renatured fusion protein TfR _ScFv-GAL4 was detected by ELISA. ⑤It was identified the characteristic of renatured fusion protein TfR (ScFv)-GAL4 binding to various tumor cell lines or tissue array of gastric cancer and breast cancer by FCM or Immunohistochemistry with antibody against GAL4, respectively.Result: â‘ The 1200bp band which was equal to its theorically calculated value could be seen in 1 % agarose gel electrophoresis when the recombinant plasmid TfR_ScFv-GAL4-pET was digested by enzyme NcoI and NotI. The TfR_ScFv-GAL4 gene sequence is accorded with the expected gene sequence by matched analysis in website: http://www.ncbi.nlm.nh.gov/blast. They showed that the recombinant expression vector TfR_ScFv-GAL4-pET was constructed successfully. â‘¡There was the most amount of TfRscFv-GAL4 fusion protein expression when the E.coli BL21 containing recombinant plasmid TfR_ScFv-GAL4-pET induced by IPTG for 4 or 6 hours, the quantity of TfR_ScFv-GAL4 fusion protein was up to 45% and 42.3% of the total bacteria protein, respectively. Expressed TfR_ScFv-GAL4 fusion protein, which is equal to theorically calculated value 46.7KD, was in cytorrhyctes, its concentration up to 1.7 mg/ml after pulification and renaturation. â‘¢The detection of ELISA showed that the GAL4 protein fragment had biological activity of responding to mouse antibody against GAL4 in the renatured fusion protein TfR ^ScFv-GAL4. â‘£The analysis of FCM suggested that TfR_ScFv protein fragment containing in the renatured fusion protein TfRscFv-GAL4 could specially binding to tumor cells expressing the TfR. Its binding rate, which is equal to the binding rate of murine antibody against TfR, ranged from 54.11% to 8.23% in 7 different kinds of human carcinoma cell lines. The observationof tissue array displayed that the renatured fusion protein TfR_ScFv-GAL4 and the murine antibody against TfR could bind to tumor tissues of gastric cancer (Positive Rate is 75.32% and 78.46%, respectively) and breast cancer (Positive Rate is 63.25% and 64.57%, respectively). There were weak response in 5 hepatic tissues, and no response in normal tissue involved in heart, spleen, adrenal cortex and blood vessel. All above suggested that the protein fragment TfR_ScFv of the renatured fusion protein TfRscFv-GAL4 displayed biology activity and specificially binding to tumor cell.Part ThreeGene Expression and its Effect Mediated by Fusion Protein TfR_scFv-GAL4 in the Targeted Tumor CellObject: To construct the expression vector containing a report gene and effector, and to study gene expression and its effect mediated by the fusion protein TfR_scFv-GAL4 in the targeted tumor cells.Method: â‘ After synthesizing recognition sequence (GAL4rec) of GAL4 DNA Binding Domain, GAL4rec was cloned into the Survivin_siRNA-pGes vector. The sequence of recombinant vector GAL4_rec-SurvivinsiRNA-pGes was identified by enzyme digestion and gene sequencing. â‘¡The renatured fusion protein TfR_ScFv-GAL4 conjugated to the recombinant vector GAL4_rec-SurvivinsiRNA-pGes and adopted molar rate were identified by 4.5 % native polyacrylamide gel electrophoresis. â‘¢The targeted complex intake mediated by the fusion protein TfR_ScFv-GAL4 and effector expression were observed through inversted fluophot. â‘£It was assayed by Hochest staining kit that tumor cell apoptosis induced by the targeted complex TfR_ScFv-GAL4-GAL4_rec-SurvivinsiRNA-pGes.Result â‘ Recombinant plasmid TfR_ScFv-GAL4-pET was not digested by enzyme EcoRI and the result show the recombinant vector inserted into recognition sequenceGAL4rec was not linearity and moved ahead. â‘¡The sequence report and matched analysis in website http://www.ncbi.nlm.nh.gov/blast suggested the expression vector of GAL4rec-Survivin_siRNA-pGes was successfully constructed. â‘¢The only one protein band could be seen in the TfR_ScFv group with different concentration of GAL4_rec-Survivin_siRNA-pGes in 4.5% native polyacrylamide gel electrophoresis, but the moved protein band was dose-dependent with addition of vector GAL4_rec-SurvivinsjRNA-pGes in GAL4 and TfR_ScFv-GAL4 group, these results showed fusion protein TfR_ScFv-GAL4 could specially bind to GAL4_rec-SurvivinsiRNA-pGes by GAL4 fragment, and the molar rate is 1: 2.5. â‘£The targeted complex system TfR_ScFv-GAL4-GAL4rec-Survivin_SiRNA-pGes and TfR_ScFv-GAL4-GAL4rec-pGes were co-cultured with 4 different kinds of human carcinoma cell lines, it was observed the green fluorescin in the tumor cell endochylema of these two groups but not in the tumor cell endochylema of the other control groups by fluorescent microscope. Also microscope could find that some adherent cells kytoplasm recovery and their bodies appeared round like the morphous of apoptosis cell in the targeted complex system TfR_ScFv-GAL4-GAL4rec-Survivin_SiRNA- pGes group, whereas the adherent cells grow well in the control groups. ⑤The targeted complex system TfRscFv-GAL4-GAL4_rec-SurvivinsiRN-pGes was co-cultured with 2 different kinds of human carcinoma cell lines, it was found that numerous apoptosis cell as well as the cells with kytoplasm recovery and round bodies by rebuilding their pictures in the targeted complex system group.Conclusion â‘ The investigation results demonstrate human-mouse chimeric antibody against TfR could remarkably inhibit the proliferation of K562 and induce its apoptosis. And one mechanism of its inducing apoptosis is through mitochondria death pathway, also involved in ferri ion transport. â‘¡The fusion protein of TfR_ScFv-GAL4 was successfully constructed and expressed, it would have biological activity after renaturation, and the function of anti-TfR_ScFv and GAL4 was not affected. â‘¢It firstlyproved that fusion protein of TfR_ScFv-GAL4 could bind to many human tumor cells, but hardly respond to normal tissues and cells, and then this suggested the fusion protein could specially bind to the tumor. (4)It firstly demonstrated the complex TfR_scFv-GAL4-GAL4rec-Survivin_siRNA-pGes could be intaken into endochylema to express the green fluorescent protein (GFP), and then induce malignant cells apoptosis. This suggested that TfR_ScFv-GAL4 has the tumor-targeted characteristic and could induce the expression of bifuction gene vector in the tumor cells.