| ObjectiveTo construct the anti-transferrin receptor monoclonal antibody targeted nanoparticles anti-TfR mAb-PEG/DIR-BOA which cargo a near-infrared fluorescent dye (DIR-BOA), study the specific combination between anti-transferrin receptor monoclonal antibody and transferrin receptor. To construct anti TfR-scFv-D4S×5, a single chain antibody with a negative tail which is provided with two biological activities as a specific and effective gene delivery system for easily conjugating DNA with single chain antibody, study the expression of the fusion protein, identify the specificity and biological activity of binding with tumor cells and the polycation polyethylenimine(PEI) and provide basis for anti-TfR antibody applications of orientation, tracing, imaging and targeted therapy on tumors.Methods1. Construction of the anti-transferrin receptor monoclonal antibody targeted nanoparticles anti-TfR mAb-PEG/DIR-BOA:①purified the anti-transferrin receptor monoclonal antibody by saturated ammonium sulfate, determined the concentration of the protein by BCA method, identified protein expression by 12% polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed the TfR binding activity by flow cytometry (FCM).②construct targeted nanoparticles by use of dual functional compound DSPE-PEG(2000)-Maleimide to connect the nanoparticles and activated anti TfR mAb with thiol group,2. Purified the nanoparticles and targeted nanoparticles by fast protein liquid chromatography(FPLC) to remove the free monoclonal antibody. 3. The anti-transferrin receptor expression of four different cells and the HDL receptor expression of two tumor cells were analyzed characterized by flow cytometry (FCM).4. The specific conjugation activity with tumor cells(HT1080) of the anti-transferrin receptor monoclonal antibody targeted nanoparticles anti-TfR mAb-PEG/DIR-BOA was analyzed by flow cytometry (FCM).5. Cloned the gene anti TfR-scFv-D4Sx5 which is more suitable for protein expression in E coli into different vector to constructed four expression vector (pET28a-6×His-scFv-D4S×5, pET28a-scFv-D4Sx5-6xHis, pGEX4T3-GST-scFv-D4S×5-8xHis, pAB1-scFv-D4S×5-6xHis) by the bioinformatics and the technology of genetic engineering.6. The positive clones were cultured and induced by isopropyl-β-D-thiogalactopy-anoside (IPTG), and identified protein expression by 12% polyacrylamide gel electrophoresis and Western-blot.7. Insoluble body and soluble protein were processed routinely, collected the purified products by Ni-NTA agrose, analyzed the products by 12% SDS-PAGE and identified by Western-blot.8. Dialysis renaturation of purification products.9. The specific conjugation activity with tumor cells(HepG2, K562) of renatured protein anti-TfR-scFv-D4Sx5 was characterized by flow cytometry (FCM).10. The transferrin receptor mediated endocytosis of renatured protein anti-TfR-scFv-D4Sx5 was observed by fluorescence microscope and the efficiency of the endocytosis was analyzed by flow cytometry (FCM)11. The binding activity between fusion protein anti-TfR-scFv-D4Sx5 and the polycation polyethylenimine(PEI) or PEI/DNA complex was identified by two separate native 5-20% gradient polyacrylamide gels with opposite electric current. Results1. By the analysis of SDS-PAGE, two bands of monoclonal antibody was observed at molecular weights 25KD and 55KD, which agreed with the theoretical value, that demonstrated the highly yield of monoclonal antibody and the concentration of the protein was 7.5mg/ml.2. The positive rate reached about 90% for the combination between monoclonal antibody products and highly expressed transferrin receptor cells HepG2 by FCM, showed the anti-transferrin receptor monoclonal antibody posses special binding activity with tumor cells which highly expressed transferrin receptor.3. FCM results showed that CHO cells nearly unexpressed TfR on surfaces, L02 cells expressed low levels and tumor cells HepG2 cells or HT1080 cells expressed great levels which were nearly up 4- to 5-fold higher than normal cells. FCM also showed that HT1080 cells expressed low levels of HDL receptor SRB I while HepG2 cells expressed at much greater levels.4. FCM data showed that the anti-transferrin receptor monoclonal antibody targeted nanoparticles anti-TfR mAb-PEG/DIR-BOA could targeted specifically to tumor cells through TfR, and such targeting can be enhanced by another targeting receptor SRB I. 5. Two bands agreeed with the theoretical value were present by two restriction endonuclease, both 1% agarose gel electrophoresis and sequencing detection identified the anti-TfR-scFv-D4Sx5 gene was successfully cloned into two different expression vectors (pET28a,pAB1).6. By the analysis of SDS-PAGE and western-blot, fusion protein 6×His-anti-TfR-scFv-D4S×5(31KD) which target gene cloned into vector pET28a was expressed as inclusion body in transformed E. Coli under 37℃induction, which then proved the expression and purification was successful. The concentration of the protein was about 2mg/ml. 7. By the analysis of SDS-PAGE and western-blot, fusion protein anti-TfR-scFv-D4S×5-6×His(31KD) which target gene cloned into vector pET28a was expressed as inclusion body in transformed E. Coli under 37℃induction, which then proved the expression and purification was successful.8. By the analysis of SDS-PAGE and western-blot, fusion protein GST-anti-TfR-scFv-D4S×5-8×His(55KD)which target gene cloned into vector pGEX4T3 was expressed as insoluble body in transformed E. Coli under 37℃induction and expressed as insoluble body and more soluble protein under 20℃induction.9. By the analysis of SDS-PAGE and western-blot, fusion protein pelB-anti-TfR-scFv-D4S×5-6×His(34KD) which target gene cloned into vector was expressed as insoluble body and little soluble protein in transformed E. Coli under 25℃induction.10. Compare anti-TfR-scFv-D4S×5 activity from different expression vector by FCM found that fusion protein 6×His-anti-TfR-scFv-D4S×5 obtained from pET28a vector showed high biological binding activity with TfR expressed on tumor cells surfaces(HepG2, K562) and the positive rate reached about 80%.11. The transferrin receptor mediated endocytosis phenomenon of renatured protein anti-TfR-scFv-D4S×5 was observed by fluorescence microscope and the efficiency of the endocytosis analyzed by FCM was about 42.6%.12. The binding activity examined by two separate native 5-20% gradient polyacrylamide gels with opposite electric current was visualized and indicated that fusion protein anti-TfR-scFv-D4S×5 could interacted with the polycation polyethylenimine(PEI) or PEI/DNA complex by electrostatic interaction.Conclusion1. The targeted nanoparticles anti-TfR mAb-PEG/DIR-BOA was successfully constructed, and showed specificity and binding activity with TfR expressed highly on the surface of HT1080 cells, it provided basis for the applications of anti TfR antibody in terms of transferrin receptor detection on cells surface, and provide basis of orientation, tracing, imaging and treatment on tumors.2. Four prokaryotic expression vector inserted with the anti-TfR-scFv-D4S×5 gene which is more suitable for protein expression in E coli were constructed successfully by the bioinformatics and the technology of genetic engineering, the fusion protein anti-TfR-scFv-D4S×5 obtained from pET28a vector showed higher expression and pronounced biological binding activity with TfR expressed on tumor cells surface and could be internalized by endocytosis, moreover, anti-TfR-scFv-D4S×5 could interacted with DNA by means of the polycation polyethylenimine(PEI). This new type of immunoporter provided a useful nonviral vector for the gene therapy and basis for anti-TfR antibody applications of orientation, tracing, imaging and targeted therapy on tumors.
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