Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.

BACKGROUND:Nowadays, gene therapy has become a new trend for disease therapy and brought promise for some refractory diseases. Its key is to choose proper cel s, genes and vectors. OBJECTIVE:To use recombinant adeno-associated virus mediatedβ-nerve growth factor (β-NGF) to transfect rat bone marrow-derived endothelial progenitor cel s in vitro, and to investigate the effect ofβ-NGF expression on the proliferation of endothelial progenitor cel s. METHODS:The endothelial progenitor cel s were isolated, cultured and identified from the bone marrow of rats. Empty vector or recombinant adenovirus-associated virus containingβ-NGF gene was transferred into endothelial progenitor cel s. We examined the transfection efficiency by fluorescence expression of green fluorescent protein. Expression ofβ-NGF protein was detected using ELISA, and its effect on the proliferation of endothelial progenitor cel s was determined using MTT method. RESULTS AND CONCLUSION:Rat endothelial progenitor cel s were isolated and cultured successful y in vitro and were identified positive by the function of cel s and immunofluorescence staining. The endothelial progenitor cel s were infected directly by the recombinant adenovirus-associated virus containingβ-NGF gene with an efficiency of 65.3%.β-NGF protein was detected in the culture supernatant of transfected endothelial progenitor cel s, which reached a high level at 10 days after gene transfection. Furthermore, there was noβ-NGF protein in the blank and empty vector groups. After transfection, the proliferative ability of endothelial progenitor cel s was increased, which was significantly higher than the blank and empty vector groups (P<0.05). But there was no difference between the latter two groups (P>0.05). These findings suggest that recombinant adenovirus-associated virus containingβ-NGF gene can be successful y transferred into rat bone marrow-derived endothelial progenitor cel s and promote the proliferation of endothelial progenitor cel s.

Aim To study the mechanism of anti-tumor effect of PHⅡ-7 to K562 and K562/A02 cells.Methods The effects of individual and combined doxorubicin on K562 and K562/A02 cells were observed by MTT assay.The coefficient of drug interaction was used to analyse the synergistic effect of PHⅡ-7,obtaining the RNA from the cells stimulated by PHⅡ-7 with different doses to analyse the MDR1 gene expression level.Finally,the cumulation of doxorubicin was observed in K562 and K562/A02 cells after being coped with PHⅡ-7.Results PH Ⅱ-7 had anti-tumor effect with IC50 of (1.37?0.37) ?mol?L-1;(1.48?0.34) ?mol?L-1 for K562 and K562/A02,respectively.It could potentiate the anti-tumor effect of dororubicin with CDI of 0.22 and 0.09 for K562 and K562/A02,respectively.PHⅡ-7 could synergistically inhibit the proliferation of K562 and K562/A02 cells.The decrease of MDR1 expression level depended on the increase of dose of PHⅡ-7 acting on cells.PHⅡ-7 could also develop the cumulation of doxorubicin in cells.Conclusion PHⅡ-7 is not only a Cytotoxinic drug but also can synergistically inhibit the proliferation of K562 and K562/A02 cells with the decrease of MDR1 expression level,especially in K562/A02 cells.

Objective: TO explore the effect of VEGF-C gene transfection on the expression of VEGF-C in human cervical carcinoma HeLa cells and the mechanisms of its anti-apoptosis effect. Methods: The con-structed pcDNA3.1(+)NEGF-C vector was transformed into human cervical cancer HeLa cells and was select-ed by G418. The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. HeLa cells with overexpression of VEGF-C were named as HeLa/S1. The expression level of NF-KB and bcl-2 mRNA was determined by RT-PCR in transfected cells. Results: After transfection by liposome, the VEGF-C mRNA level and the expression of VEGF-C protein in transfected cells were higher than those in the control groups. HeLa/S1 cell line was successfully established. In HeLa/S1 cells, the expression of NF-κB (2.06±0.09 vs 1.35±0.02 vs 1.38±0.02 P<0.05) and bcl-2 gene mRNA (2.02± 0.67 vs 0.41±0.06 vs 0.37±0.06, P<0.05) level were higher than those in the control groups. Conclusion: VEGF-C gene transfection by liposome can increase the expression of VEGF-C in human cervical cancer HeLa cells. NF-κB is stimulated and induces the overexpression of bcl-2 gene in HeLa/S1 cells.

Aim To investigate the potential suppressing effect of calmodulin antagonist EBB on metastasis-associated properties of human metastatic ovarian clear cell adenocarcinoma cell ES-2.Methods MTT assay was used to assess the growth inhibition of EBB on ES-2 cells.The invasive capacity and motility potential were determined by Transwell chamber assay and Wound assay.[Ca2+]i was observed under the laser scanning confocal microscopy.Results EBB inhibited the proliferation of ES-2 cells in vitro.The IC50 on ES-2 cells was(13.67?1.56)?mol?L-1.The invasive ability and motility potential of ES-2 cells were decreased after exposure to 3,7 and 14 ?mol?L-1 EBB respectively(P

Animals ; Cell Division ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Growth Inhibitors ; pharmacology ; Hepatocytes ; cytology ; drug effects ; Lamiaceae ; chemistry ; Rats

Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab' expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was induced to express in E. coli 16C9. The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.
4-1BB Ligand ; biosynthesis ; genetics ; Antibodies, Bispecific ; immunology ; Antigens, CD20 ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; Immunotherapy ; methods ; Lymphoma, Non-Hodgkin ; therapy ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo study the relationship between soluble resistance-related calcium-binding protein (sorcin) gene and multidrug resistance gene (mdr1), and their significance in clinical drug resistance and prognosis of acute myeloid leukemia (AML).

METHODSAmplification of sorcin gene and mdr1 gene in K562/A02 cell detected by Northern blot, were monitored by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) in 65 AML patients and 27 normal controls, with their relationship and clinical outcame analyzed.

RESULTSThe amplification of sorcin gene and mdr1 gene in AML patients were significantly higher than that in the normal control, which were related to clinical drug resistance and prognosis. The amplification of sorcin gene was related to the amplification of mdr1 gene in the two groups. The clinical drug resistance incidence rate and complete remission rate were 92.9% and 7.1% in sorcin(+)/mdr1(+) group. They were 8.6% and 91.4% in the sorcin(-)/mdr1(-) group (P < 0.001).

CONCLUSIONThe co-amplification of sorcin and mdr1 gene can be taken as a good indicator of clinical drug resistance and prognosis of AML.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Acute Disease ; Blotting, Northern ; methods ; Calcium-Binding Proteins ; genetics ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Expression ; Humans ; K562 Cells ; Leukemia, Myeloid ; genetics ; physiopathology ; Neoplasm Proteins ; genetics ; Prognosis

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Solubility ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-1 ; genetics ; isolation & purification ; metabolism