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1.
Invest New Drugs ; 42(1): 116-126, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38253746

RESUMO

Nivolumab can cause fatal myocarditis. We aimed to analyze the clinical characteristics of nivolumab-induced myocarditis and provide evidence for clinical diagnosis, treatment, and prevention. Studies involving nivolumab-induced myocarditis were identified in electronic databases from 2000 to 2023 for retrospective analysis. A total of 66 patients were included, with a median age of 68 years. The median onset time of myocarditis is 11.5 days. The main organs affected in persons presented with myocarditis are heart (100.0%) and skeletal muscle (22.7%). The main clinical manifestations are dyspnea (49.2%), fatigue (47.6%), and myalgias (25.4%). The levels of troponin, troponin T, troponin I, creatine kinase, creatine kinase myocardial band, creatine phosphokinase, C-reactive protein, brain natriuretic peptide, and N-terminal brain natriuretic peptide precursor were significantly increased. Histopathology often shows lymphocyte infiltration, myocardial necrosis, and fibrosis. Myocardial immunological parameters usually present positive. Cardiac imaging often suggests complete heart block, intraventricular conduction delay, arrhythmia, myocardial infarction, edema, left ventricular ejection fractions reduction, ventricular dysfunction, and other symptoms of myocarditis. Forty-two (63.6%) patients achieved remission within a median time of 8 days after discontinuation of nivolumab and treatment with systemic corticosteroids, immunoglobulins, plasmapheresis, and immunosuppressant. Thirty-five patients eventually died attributed to myocarditis (68.6%), cancer (20.0%), respiratory failure (5.7%), and other reasons (5.7%). Nivolumab-induced myocarditis should be comprehensively diagnosed based on clinical symptoms, histopathological manifestations, immunological parameters, and cardiac function imaging examinations. Nivolumab should be discontinued immediately, plasmapheresis and systemic corticosteroids combined with immunoglobulins or immunosuppressants may be an effective treatment.


Assuntos
Antineoplásicos Imunológicos , Miocardite , Humanos , Idoso , Nivolumabe/efeitos adversos , Miocardite/induzido quimicamente , Miocardite/diagnóstico , Miocardite/terapia , Antineoplásicos Imunológicos/efeitos adversos , Estudos Retrospectivos , Peptídeo Natriurético Encefálico/efeitos adversos , Imunossupressores/uso terapêutico , Corticosteroides/efeitos adversos , Creatina Quinase
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 26(2): 576-583, 2018 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-29665935

RESUMO

OBJECTIVE: To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. METHODS: PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2+-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin-Scal-1+c-Kit+ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. RESULTS: The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. CONCLUSIONS: A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.


Assuntos
Hematopoese , Animais , Tetracloreto de Carbono , Escherichia coli , Células-Tronco Hematopoéticas , Ligantes , Camundongos , Receptores Notch , Transdução de Sinais
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 24(3): 827-32, 2016 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-27342518

RESUMO

OBJECTIVE: To investigate the microRNA (miRNA) expression in plasma of patients with aGVHD and without aGVHD after allo-hematopoietic stem cell transplantation (allo-HSCT). METHODS: The miRNAs (miR-423, mirR199a-3p, miR93*, miR377) expression levels in peripheral blood plasma of 25 patients before and after allo-HSCT were detected by real-time PCR. RESULTS: miR-423, miR199a-3p and miR-93* in aGVHD group were significantly upregulated (P<0.05); miR-377 expression was not significantly different between aGVHD and non-aGVHD (P>0.05). CONCLUSION: The expression of miR-423, miR-199a-3p, miR-93* are upregulated in aGVHD group, which can be used as biomarkes to monitor and to diagnose aGVHD.


Assuntos
Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas , MicroRNAs/sangue , Biomarcadores/sangue , Doença Enxerto-Hospedeiro/sangue , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
4.
Sci Rep ; 6: 26003, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27188577

RESUMO

Physical and chemical insult-induced bone marrow (BM) damage often leads to lethality resulting from the depletion of hematopoietic stem and progenitor cells (HSPCs) and/or a deteriorated BM stroma. Notch signaling plays an important role in hematopoiesis, but whether it is involved in BM damage remains unclear. In this study, we found that conditional disruption of RBP-J, the transcription factor of canonical Notch signaling, increased irradiation sensitivity in mice. Activation of Notch signaling with the endothelial cell (EC)-targeted soluble Dll1 Notch ligand mD1R promoted BM recovery after irradiation. mD1R treatment resulted in a significant increase in myeloid progenitors and monocytes in the BM, spleen and peripheral blood after irradiation. mD1R also enhanced hematopoiesis in mice treated with cyclophosphamide, a chemotherapeutic drug that induces BM suppression. Mechanistically, mD1R increased the proliferation and reduced the apoptosis of myeloid cells in the BM after irradiation. The ß chain cytokine receptor Csf2rb2 was identified as a downstream molecule of Notch signaling in hematopoietic cells. mD1R improved hematopoietic recovery through up-regulation of the hematopoietic expression of Csf2rb2. Our findings reveal the role of Notch signaling in irradiation- and drug-induced BM suppression and establish a new potential therapy of BM- and myelo-suppression induced by radiotherapy and chemotherapy.


Assuntos
Medula Óssea/fisiologia , Predisposição Genética para Doença , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lesões Experimentais por Radiação/fisiopatologia , Receptores de Interleucina-3/metabolismo , Animais , Células Sanguíneas , Medula Óssea/efeitos da radiação , Proteínas de Ligação ao Cálcio , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Masculino , Camundongos Endogâmicos C57BL , Células Progenitoras Mieloides/fisiologia , Regeneração , Transdução de Sinais , Baço/citologia , Regulação para Cima
5.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 22(3): 807-11, 2014 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-24989299

RESUMO

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Escherichia coli/metabolismo , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Serrate-Jagged
6.
BMC Cancer ; 14: 463, 2014 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-24952875

RESUMO

BACKGROUND: Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear. METHODS: Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software. RESULTS: In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression. CONCLUSIONS: Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.


Assuntos
Apoptose/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas Musculares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Motivos de Aminoácidos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Progressão da Doença , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 19(6): 1415-8, 2011 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-22169294

RESUMO

The purpose of this study was to compare the efficacy of CEP plus G-CSF and CVP plus G-CSF regimens in the mobilization and collection of peripheral blood hematopoietic stem cells (PBHSC), and in the hematopoietic recovery. 57 patients with non-Hodgkin's lymphoma (NHL) underwent autologous PBHSC transplantation were analyzed retrospectively. The PBHSC were mobilized and collected by using CEP plus G-CSF and CVP plus G-CSF respectively, and were retransfused into these NHL patients after preconditioning, then the mobilization efficacy, adverse reactions and hematopoietic recovery were analyzed. The results showed that the WBC count decreased to ≤ 1.0 × 10(9)/L, platelet amount dropped to ≤ 40 × 10(9)/L during peripheral blood stem cell mobilization of all patients, which indicated successful collection of PBHSC. The mean value of (4.38 ± 3.40) × 10(8)/kg mononuclear cells (MNC) containing (2.79 ± 2.53) × 10(6)/kg CD34(+) cells were collected in CEP plus G-CSF group, while the mean value of (3.31 ± 1.23) × 10(8)/kg MNC containing (2.02 ± 0.87) × 10(6)/kg CD34(+) cells were collected in CVP plus G-CSF group. The efficacy of mobilization in CEP plus G-CSF group was significantly higher than that in CVP plus G-CSF group (p < 0.05). After preconditioning, bone marrow was suppressed in all patients. The average time of WBC count recovery to ≥ 1.0 × 10(9)/L was 11.4 days in CEP plus G-CSF group and 12.3 days in CVP plus G-CSF group; the average time of platelet amount recovery to ≥ 50 × 10(9)/L was 18.6 days in CEP plus G-CSF group and 19.3 days in CVP plus G-CSF group. The statistical analysis showed no significant difference in the average time of hematopoietic recovery between 2 groups. It is concluded that autologous PBHSC transplantation shows significant effect for treatment of patients with NHL. Either modified CEP or CVP plus G-CSF regimen is safe and effective in PBHSC mobilization. The CEP plus G-CSF regimen is better than CVP plus G-CSF regimen.


Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Linfoma não Hodgkin/terapia , Transplante de Células-Tronco de Sangue Periférico , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Transplante Autólogo , Adulto Jovem
8.
Mol Cell Biochem ; 340(1-2): 7-14, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20157766

RESUMO

The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.


Assuntos
Proliferação de Células , Leucemia Promielocítica Aguda/metabolismo , Receptores Notch/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HL-60 , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição HES-1 , Transfecção
9.
Leuk Res ; 33(6): 798-802, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18937977

RESUMO

The evolutionarily conserved Notch signaling pathway plays a pivotal role in cell proliferation, apoptosis, and cell fate decision from invertebrates to vertebrates, and is oncogenic in some human hematopoietic malignancies. To study the role of Notch signaling in B-lymphoma, we expressed a soluble fragment of human Delta-like1 (hDll1) in E. coli, which was shown to activate the Notch signaling. Incubation of Burkitt's lymphoma Raji cells with the soluble hDll1 led to gamma-secretase-dependent up-regulation of a Notch downstream gene, Hes1. This treatment synergized with B-cell receptor (BCR)-mediated signaling to promote proliferation of Raji cells in vitro, which was cancelled by GSI. We further showed that Notch signaling significantly repressed, while gamma-secretase inhibitor (GSI) enhanced, "natural" apoptosis of Raji cells. Because c-myc is a downstream gene of both Notch signaling and BCR signaling, and GSI blocked c-myc expression in the presence of hDll1 and anti-IgM, Notch signaling might interact with BCR signaling at the level of c-myc expression to regulate proliferation and apoptosis of B-lymphoma cells.


Assuntos
Proliferação de Células , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 16(5): 1227-31, 2008 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-18928634

RESUMO

Ex vivo expansion of hematopoietic progenitor cells (HPCs) is valuable for clinical application, however, traditional ex vivo culture negatively affects long-term hematopoietic reconstitution ability. In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported. Active Notch signal inhibits the differentiation of HSCs while promotes their expansion, suggesting that ex vivo expansion of hematopoietic progenitor cells could be enhanced by manipulating Notch signal pathways. In this article the Notch signal pathways, Notch signal and maintenance of hematopoietic progenitor cells, Notch signal and expansion of hematopoietic progenitor cells and molecular mechanism of Notch signal maintaining undifferentiation of hematopoietic progenitor cells were reviewed.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Transdução de Sinais
11.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 16(4): 910-4, 2008 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-18718088

RESUMO

In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.


Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Vetores Genéticos/genética , Fragmentos Fc das Imunoglobulinas/biossíntese , Imunoglobulina G/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Membrana/biossíntese , Pichia/metabolismo , Proteínas de Ligação ao Cálcio/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Pichia/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Serrate-Jagged , Transfecção
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 24(5): 450-2, 2008 May.
Artigo em Chinês | MEDLINE | ID: mdl-18466699

RESUMO

AIM: To construct a Pichia pastoris expression vector PIC- hDll1(ext)-Fc, and to express the fusion protein containing the extracellular domain of human Delta-like1 and the Fc fragment of human IgG1 fusion gene in Pichia pastoris. METHODS: The extracellular domain of human Delta-like1 gene was amplified from pEF-BOSneo-hdll1(ext)-Fc by PCR. The expression vector was constructed by DNA recombination. The constructed plasmid was transformed into yeast GS115 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of protein was induced by addition of methanol.Then analyzed protein expression by SDS-PAGE and Western blot. RESULTS: The extracellular domain of human Delta-like1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hDll(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. CONCLUSION: The hDll1(ext) gene has been successfully cloned and expressed in the form of Fc fusion protein, which provides a new fusion protein for further experiments.


Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Pichia/genética , Proteínas Recombinantes/farmacologia , Eletroporação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vetores Genéticos/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Metanol/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão
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