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Objective:To preparation TREM2-specific monoclonal antibodies and to evaluation their properties and applica-tions lay a foundation for the development of druggable antibodies against TREM2 targets.Methods:The GST fusion protein containing the extracellular segment of TREM2 was expressed and purified,which was used as antigen to immunize mice.The mouse with high antiserum titer was used to generate specific monoclonal antibodies against TREM2 through cell fusion and monoclonal screening.The specificity and affinity of the monoclonal antibodies and their applications in different immunological experiments were detected.Results:A total of 29 strains of anti-TREM2 monoclonal antibodies were obtained,of which 24 strains could specifically bind to TREM2.The EC50 of antibodies were calculated above nanomolar.These monoclonal antibodies could be used for specific detection of TREM2 in Western blot,immunoprecipitation,flow cytometry and immunofluorescence experiments.Conclusion:Monoclonal anti-bodies against TREM2 with high affinity and specificity are successfully generated,which lay the foundation for the research and the development of antibody drugs targeting TREM2.
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Gastrointestinal cancer is one of the most common malignant tumors in China which affects people health seriously. Along with the investigation of the cancer molecular markers and the application of novel target agents, the therapeutic modalities for gastrointestinal cancers have been changing gradually. More and more target drugs, such as trastuzumab, cetuximab, bevacizumab,have been used in clinical practice. The individualized therapy based on the molecular classification will benefit more patients and is the inevitable trend of gastrointestinal cancer treatments in the future.
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Humanos , Biomarcadores de Tumor , Neoplasias Gastrointestinales , Metabolismo , Terapéutica , Medicina de PrecisiónRESUMEN
Phage display random heptapeptide library was screened with recombinant P37 in this study. The positive phage clones were identified by ELISA and were sequenced, and the amino acid sequences of the polypeptides displayed on phage were deduced. After GST-polypeptides fusion protein was constructed and expressed, its binding to P37 was determined by GST-pull down and Western blot. After 4 rounds of bio-panning, the enriched positive phage clones were identified by ELISA. Eighteen positive phage clones were sequenced and the peptide sequences were as follows. ACAPKPPWLC (12/18), RPLSIDPWSPHL (3/18), RPLSNDPWSPHL (1/18), QNMMSPIEGVRI (1/ 18) and WAPEKDYMQLMK (1/18). The results from GST-pull down and Western blot showed that peptide RPLSIDPWSPHL could interact with P37. The study will be helpful for identifying the protein reacting with P37.
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Animales , Proteínas Bacterianas , Metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Mycoplasma hyorhinis , Metabolismo , Oligopéptidos , Metabolismo , Biblioteca de Péptidos , Unión Proteica , PorcinosRESUMEN
Hsc70-SNCG fusion protein cDNA fragment containing signal peptide sequence of Igkappa, MMP9, and P37 was inserted into the vector pVAX1 to construct recombinant plasmid pVAX-Igkappa-Hsc70-SNCG, pVAX-MMP9-Hsc70-SNCG, and pVAX-P37-Hsc70-SNCG. Three eukaryotic vectors were constructed and verified by restriction enzyme digestion and sequencing. After transfection with recombination plasmids in QM-7 cells, the transient expression and secretion of three fusion proteins were detected by ELISA and Western Blot. The results suggested that Hsc70-SNCG carrying three different signal peptides could be expressed and secreted by transfected cells, and three signal peptides effectively directed secretion of fusion protein by QM-7 cells. BALB/c mice were immunized by three plasmids using gene gun system. The serum levels of anti-SNCG antibodies in mice were measured by ELISA. The results showed that three secreted plasmids could stimulate humoral immune responses to SNCG in mice, which depended on the secreted expression levels induced by signal peptides.
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Animales , Humanos , Ratones , Vectores Genéticos , Inmunoglobulina G , Ratones Endogámicos BALB C , Proteínas de Neoplasias , Genética , Alergia e Inmunología , Plásmidos , Metabolismo , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , Transfección , gamma-Sinucleína , Genética , Alergia e InmunologíaRESUMEN
Previous work showed that there was high ratio of Mycoplasma hyorhinis infection in human gastric carcinoma. P37 protein is a membrane lipoprotein of Mycoplasma hyorhinis. It was reported that P37 inhibited cell adhesion and induced tumor invasiveness. To investigate the function of P37 in cancer development, the p37 gene was subcloned into shuttle vector of pAdTrack-CMV. Linearized pAdTrack-CMV-p37 was co-transformed into BJ5183 cells with adenoviral genomic plasmid pAdEasy-1. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From the supernatant and cell lysis, the presence of recombinant adenovirus P37 was proved by PCR. And then BICR cells with this recombinant adenovirus of P37 were successfully infected. RT-PCR and Western blot demonstrated the transcription and expression of P37 in infected BICR cells. And the soluble P37 protein can be secreted into the culture supernatant of infected BICR cells. In the migration assay in vitro, the number of migration BICR cells infected with Ad-p37 was 52.03% more than that of control. The construction of recombinant adenovirus provided a good tool for studying P37 function and its molecular mechanism, which will be further used for in vivo migration and invasion experiments.
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Objective:To investigate the effects of Adp53 and F56 on the growth and lung metastasis of breast cancer.Methods:The BICR-H1 cells were inoculated into the mammary fatty pad of BALB/C nude mice and NOD/SCID mice to establish breast cancer model.Then the nude mice with xenograft tumor were randomized into group Adp53+F56,Adp53,F56 and control.The NOD/SCID mice with xenograft tumor were randomized into group Adp53+F56,Adp53,F56,Adlacz and control.They were theated for 3 weeks according to the plan,diversity of the volume and histopathology of xenograft tumor of nude mice was observed and the expressions of p53 and VEGF gene,and microvessel density(MVD)were detected by immunohistochemistry.Lung metastasis of breast cancer in NOD/SCID mice was observed.Results:(1)Intratumoral injections of Adp53,F56,and their combination resulted in an inhibition on the growth of xenograft tumor of BICR-H1 cells.The ultimate relative growth volumes of groups Adp53+F56,Adp53,F56 and control were 2.47,4.37,4.69 and 12.49 respectively.(2)After treatment,P53 positive rate of group Adp53+F56,Adp53 increased 9.4%,6.3% than before respectively,but compared with control group,the difference is not significant(P=0.693);VEGF protein of group Adp53+F56,Adp53 and F56 decreased 21.9%,9.4% and 3.1% than before respectively,but compared with control group,the difference was not significant(P=0.284).Necrosis and decrease of vessel in the tumor and morphological change of endothelium were observed under light microscope in the groups Adp53+F56,Adp53 and F56.MVD estimated by FⅧ-RA staining of group Adp53+F56,Adp53 and F56 were 14.50?2.54,16.28?3.44 and 18.06?7.66,compared with control group(24.93?6.53),the difference is significant(P=0.000).(3)The average number of lung metastasis of NOD/SCID mice in group Adp53+F56,Adp53 and F56 were 1.143?0.378,2.750?0.886 and 3.375?0.518 respectively,lower than Adlacz group(5.000?0.816)and control group(5.670?0.817)obviously(P=0.000).Conclusion:Adp53 combined with F56 can greatly inhibit growth and matastasis of breast cancer in vivo.The mechanism of anti-tumor effects of Adp53 and F56 may be related to the anti-angiogenesis effect on malignant tumor through inhibiting the expression and activity of VEGF.
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Objective: Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185 erbB2 is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185 erbB2 monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185 erbB2 chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr - cell. Methods: The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr - cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185 erbB2 chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185 erbB2 , were measured with MTT assay in vitro. Results: The anti-p185 erbB2 chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr - was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185 erbB2 specifically, but did not react with that non-overexpressing p185 erbB2 . Immunoprecipitation test confirmed that the chimeric antibody could bind to p185 erbB2 specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185 erbB2 . Conclusion: The anti-p185 erbB2 mouse/human chimeric antibody that was expressed in CHO-dhfr - cells can bind to p185 erbB2 specifically and inhibit proliferation of SKBR3 cells overexpressing p185 erbB2 . It has a potential application in biotherapy of cancer.
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Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.