Therefore, the in vitro cytotoxicity of 7G4\1\Ga to CT83\expressing cells was evaluated. were expressed in lung cancer, stomach cancer, colorectal cancer, urothelial cancer, cervix cancer, breast cancer, and various tumor cell lines (U\266/70, HeLa, U\266/84, K\562, U\2OS). Hence, CT83 may be a potential target for antibody\photosensitizer conjugate\based PDT to a variety of malignancies. Herein, we developed and characterized a novel mouse monoclonal antibody specific against human CT83 (CT83 mAb 7G4), which can effectively bind to 1\Ga to form antibody\photosensitizer complex 7G4\1\Ga. The enzyme\linked immunosorbent assay (ELISA), flow cytometry, and cytotoxicity activity assays revealed that 7G4\1\Ga was able to recognize human CT83 with high specificity. Furthermore, 7G4\1\Ga showed greater cytotoxicity to CT83\expressing human cancer cells in vitro than that of 1\Ga. These results suggest that the antibody\conjugated photosensitizer, anti\CT83 mAb and 1\Ga, might be a promising targeted PDT for effective treatment of CT83\expressing Rabbit Polyclonal to STEA3 tumors. 2.?MATERIALS AND METHODS 2.1. Materials Freund’s complete and incomplete adjuvants, HAT (hypoxanthine, aminopterin, thymidine) medium, and polyethylene glycol 1500 (PEG1500), were purchased from Sigma\Aldrich (St. Louis, MO, USA). Soluble human CT83 protein prokaryotic expression system (pET28a\CT83 in BL21 (DE3)) was constructed in our laboratory. The SBA Clonotyping (IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, , ) System\HRP kit was purchased from Southern Biotech (USA). Gallium(III) 5,10,15\tris (ethoxycarbonyl) corrole (1\Ga) was obtained from Key Laboratory of Functional Molecular Engineering of Guangdong Province, South China University of Technology, and prepared according to the literature.3 6?weeks old, female BALB/c mice were purchased from the Guangdong Animal Research Institute (Guangzhou, China). All animals were strictly handled according to the Good Animal Practice Requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The present study was approved by the Animal Ethics Committee of the Dongguan Key Laboratory of Medical Bioactive Molecular Developmental and Translational Research, Guangdong Medical University (Approval no. 20150007). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Human colorectal cancer cell lines (RKO, SW1116), MRX-2843 lung cancer cell line (NCI\H1299), human hepatoma cell line (HuH\7), cervix cancer cell line (HeLa), breast cancer cell lines (MCF\7, MDA\MB\231), nasopharyngeal cancer cell lines (HNE\1, CNE\2), embryonic kidney cell line (293T) and Sp2/0\Ag14 cell MRX-2843 line were obtained from the Cell bank of Chinese Academy of Sciences (Shanghai, China). Human lung cancer, nasopharyngeal cancer, cervix cancer, colorectal cancer, breast cancer, gastric cancer, melanoma, urothelial carcinoma, and endometrioid adenocarcinoma tissue sections were acquired from the Fifth People’s Hospital of Dongguan under an approved IRB and informed consent was obtained from all human subjects before the study. 2.2. CT83 epitope prediction According to CT83 primary structure reported from the NCBI Guide at http://www.ncbi.nlm.nih.gov, the secondary structure, surface probability, flexibility, and antigenicity of the CT83 were analyzed by DNAStar software. Amino acid sequences with \turn or random\coil, and few \helix and \sheet, good hydrophilicity, high accessibility, high flexibility, and strong antigenicity were selected as protein antigen epitope sequences. 2.3. Synthesis of genes encoding CT83 antigen fragments According to the CT83 epitope prediction results, the best epitope sequence was selected and synthesized by gene synthesis technology. The synthesized gene sequence also was amplified by PCR. The primers for PCR were as follows: 5\CTGGATCCTATCGTCGTTTTCAGCGAAAC\3 (forward); 5\GCCTCGAGTTAGGTACTTTTACGATGCG\3 (reverse). Conditions for PCR amplification were 5?minutes predenaturation at 94C and followed by 30 cycles of 30?seconds denaturation at 94C, 30?seconds annealing MRX-2843 at 54C and 30?seconds extensions at 72C, and for 5?minutes at 72C for final extension. The amplified gene sequence was inserted into the pET28a vector at restriction enzyme sites BI. 2.4. Preparation of recombinant human CT83 proteins The plasmid pET28a vector and the target gene were digested by corresponding endonucleases BI, respectively. The fragments were ligated by T(4) DNA ligase to gain recombinant expression vector. Then, the recombinant plasmid pET28a\CT83 was electro\transformed into BL21 (DE3) strain, and expressed in the bacteria under induction of 0.5?mmol/L isopropyl \D\thiogalactoside (IPTG), at 20C for 12?hours. After this, the bacteria were centrifuged at 5000?CT83 only.
Therefore, the in vitro cytotoxicity of 7G4\1\Ga to CT83\expressing cells was evaluated