Construction of eukaryotic vector of monkey B virus glycoprotein D gene and the gD gene expression / 中国比较医学杂志

Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Synthesis and eukaryotic expressing of the gene encoding extracellular domains of gB protein B virus / 中国比较医学杂志

Objective To get the gene encoding extracellular domains of gB protein of B virus and analyze its expression in the eukaryocyte cell.Methods synthesizing gene fragment encoding extracellular domains of gB protein of B virus was by using synthesis gene, then digested with the restriction endonucleases BamHⅠand NotⅠand inserted into eukaryotic expressing vector pEGFP-N3.pEGFP-N3-GB合 was transfected into 293 cells.After protein extraction, the expression of gene was detcted by western blotting, and the cellular localization of the gene was analyzed by immunofluorescence and laser scanning confocal microscopy.Results pEGFP-N3-GB合were expressed in 293 cells and on the cell membrane.Conclusion eukaryotic expressing system can produce specific antigen recombination protein of B virus gB protein and express on the cell membrane.

Expression and role of ubiqutin carboxyl-terminal hydrolase L1 in lens with age-related cataract under oxidative stress / 中华实验眼科杂志

Background Oxidative damage is a major cause of age-related cataracts,and the ubiquitinproteasome system is involved in lens differentiation and development.Ubiquitin carboxy-terminal hydrolase L1 (UCHL1),one of key enzymes of ubiquitin-proteasome system,was discovered to participate in the age related diseases and oxidative stress damage. Objective This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods Lens capsule were collected from 24 patients with age-related cataract(including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery.Five normal lens capsule membranes were obtained from eye bank of Tongji University.Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study.Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology.UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome,and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group.UCHL1 over-expressing cells were then exposed to different concentrations (0.2,0.3,0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer,but significantly different expressing levels were seen among normal lens capsular membrane,cortical cataract and nuclear cataract ( F =13.411,P =0.000),and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P =0.000,P =0.000).No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract ( P =0.164).Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group,illuminating a successful transfection of UCHL1 in SRA01/04 cells.MMT assay revealed that the A570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions UCHL1 has an antioxidative ability,and it might plays an important role in the progress of age-related cataract.

Construction of granulocyte-macrophage colony stimulating factor gene eukaryotic expressing plasmid and identification of its biological activity / 实用儿科临床杂志

Objective To construct mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) gene eukaryotic expressing plasmid pcDNA3-GM-CSF, to transfect the recombinant into erythroleukemia cell line FBL-3, and identify their biological activity.Methods GM-CSF gene eukaryotic expressing plasmid was constructed by subclone and recombinant was transfected into FBL-3 cells by electroporation. After screening by G418 and cloning by limiting dilution,we obtained positive cell clones(FBL-3-GM-CSF). PCR and RT-PCR were used to identify the integration and stable expression of GM-SF gene in FBL-3-GM-CSF cells. The biological activity was confirmed by the hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. Results Mouse GM-CSF cDNA was amplified from the prokaryotic expressing plasmid PET-30a(+)-GM-CSF by PCR firstly and BamH Ⅰ and EcoRⅠrestriction sites were introduced. The inserted fragment was cut by BamH Ⅰ and EcoR Ⅰ digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleared with appropriate endoneucleases and sequenced. The findings showed that the orientation of the insert was correct, while no rearrangement or mutation was found. PCR and RT-PCR assay showed that GM-CSF gene had integrated into FBL-3-GM-CSF cells and stably expressed. The hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay demonstrated that the cultured supernatant of FBL-3-GM-CSF cells of expressing GM-CSF should obviously stimulate proliferation of murine marrow mononuclear cells, and could stimulate hematopoietic progenitor cell colony formation. The number of colony formation was 54.67?4.83. The rate of colony formation was 0.547 %.Conclusions GM-CSF gene eukaryotic expressing plasmid is constructed successfully. A cell clone, which can express stably GM-CSF gene and possess biological activity,is obtained. Our studies have founded the base for the preparation of GM-CSF gene-modified vaccine of tumor cell and the study of feasibility of immune therapy of leukemia.

Expression on dhfr-CHO cells and activity study of CD137 (human 4-1BB) / 中国免疫学杂志

Objective:To set up a eukaryotic system for high expressing human CD137 and to investigate the role of CD137 and CD137L on signals transduction of cells.Methods:The pCDNA3 plasmid containing full length of human CD137 cDNA sequence(CMV ILA SEN,CIS) and pSV2 dhfr plasmid were cotransfected into dhfr CHO cells by lipoid mediating method. The positive clone was selected with G418. Expression of CD137 on dhfr CHO cells were induced by MTX and detected by RT PCR, immunocytochemistry and flow cytometry.It's activity study was done by method of incorporating 3H TdR.Results:CD137 expressed on the surface of dhfr CHO, expression rate was 96.07%, it's activity study indicated that CD137 increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.Conclusion:dhfr CHO cells that highly express CD137 were established. CD137 can increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.

Construction of Sense and Antisense of Human Heparanase and Green Fluorescent Protein Eukaryotic Co-expressing Vectors and Their Expression in Pancreatic Cancer Cell Lines SW1990 / 昆明医科大学学报

Objective To construct sense and antisense of human heparanase and green fluorescent protein eukaryotic expressing vectors and then transfect these vectors into Pancreatic Cancer Cell Lines SW1990.Methods The human Heparanase cDNA fragment contained in the pcDNA3 Hpa vector was cloned into the enhanced green fluorescent protein eukaryotic expressing vector pIRES2-EGFP in cis-direction or trans-direction.The recombinant vectors were identified by digestion of BamH I and further identified by DNA sequencing.The recombinant were transfected to pancreatic cancer cells SW1990 by liposome method.After 24h,the transfected cells were observed under fluorescent inverted microscope.Results After digested by BamH I,two fragments with the length of 5.3 kb and 1.7 kb were formed in sense fluorescent eukaryotic expressing vector,while two other fragments with the length of 6.5 kb and 0.5 kb were formed in antisense fluorescent vector.The DNA sequencing was also confirmed the linking direction of sense and antisense recombinant.Green fluorescence of the transfected cells could be observed under fluorescent inverted microscope after 48h of transfection.Conclusion Human heparanase sense and antisense fluorescent eukaryotic expressing vectors are successfully constructed and transfected to human pancreatic cancer cell lines.

Establishment of HEK293 cell line expressing sense and antisense Alu-Sx stably and efficiently / 第三军医大学学报

Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.

The study on expression and activity of human mutant CD59 gene / 中国免疫学杂志

Objective: To amplify two human mutant CD59 eukaryotic expressing systems and investigate mutant CD59 functional activity. Methods: Mammalian expression vector PLATER of mutant CD59 cDNAs was transfected into CHO together with the pcDNA by lipofectamine,which confered resistance to G418(400 ?g/ml). The positive clones were tested by FIH. Activity of both mutants CD59 was determined by BCECF release assay. Results: Mutant CD59 cDNAs subcloned into the mammalian expression vector PLATER and transfected CHO together with the pcDNA,which confered resistance to G418. The positive clones were tested by FIH.Activity of both mutants CD59 before and after glycation was determined by BCECF release assay,both of them could restrict MAC formation ,and glycation could inhibit CD59. Conclusion: A eukaryotic system that expressing mutant CD59 cDNA was successfully set up.It was found that mutant CD59 could restrict MAC formation,and glycation could inhibit mutant CD59. These would be helpful for the furthur study of link mutant CD59 and the vascular proliferative of diabetes.