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1.
Article in Chinese | WPRIM | ID: wpr-307962

ABSTRACT

<p><b>OBJECTIVE</b>To compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method.</p><p><b>METHODS</b>Four recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed.</p><p><b>RESULTS</b>No significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml).</p><p><b>CONCLUSION</b>GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.</p>


Subject(s)
Adenoviridae , Metabolism , Physiology , Capsid Proteins , Metabolism , DNA, Viral , Green Fluorescent Proteins , Metabolism , Real-Time Polymerase Chain Reaction , Methods , Viral Plaque Assay , Methods , Virus Replication
2.
Article in Chinese | WPRIM | ID: wpr-307901

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression characteristics of Gluc-Fluc dual luciferase plasmid after its transfection into MB49 bladder cells.</p><p><b>METHODS</b>pAAV2neoCAG-Gluc-2A-Fluc and pAAV2neo-Gluc plasmids were separately transfected into MB49 cells via LipofectamineTM2000. The Gluc activity in the cell culture supernatant and the Fluc activity in the cells were detected by luminometer and Lumina Imaging system.</p><p><b>RESULTS</b>The luminometer result showed that the activity of Gluc in the supernatant increased gradually in a cell number- and time-dependent manner, while Fluc activity in the cells increased with the cell number but not with time. The Lumina Imaging system showed that Gluc-Fluc was successfully expressed in MB49 bladder cells and cell lines with stable Gluc-Fluc expression were obtained after G418 selection.</p><p><b>CONCLUSION</b>Gluc in the dual luciferase plasmid retains its expression characteristics. Due to the advantages of Fluc in localization in living imaging and the easy quantitative detection of Gluc, the dual luciferase plasmid, after transfection in MB49 bladder cells, allows reliable and dynamic detection of tumor growth in animal models.</p>


Subject(s)
Animals , Cell Line, Tumor , Genetic Vectors , Luciferases , Genetics , Metabolism , Mice , Plasmids , Genetics , Metabolism , Transfection
3.
Article in Chinese | WPRIM | ID: wpr-235121

ABSTRACT

<p><b>OBJECTIVE</b>To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.</p><p><b>METHODS</b>Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).</p><p><b>CONCLUSION</b>The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.</p>


Subject(s)
Amino Acid Sequence , Animals , Antibodies , Genetics , Allergy and Immunology , Antibody Specificity , Arthritis, Rheumatoid , Allergy and Immunology , Cell Surface Display Techniques , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , HEK293 Cells , Humans , Immunoglobulin G , Genetics , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin kappa-Chains , Genetics , Lymphocytes , Allergy and Immunology , Metabolism , Molecular Sequence Data , Peptide Library , Recombinant Proteins , Genetics , Allergy and Immunology , Transfection
4.
Article in Chinese | WPRIM | ID: wpr-289994

ABSTRACT

<p><b>OBJECTIVE</b>To construct human renal cell carcinoma patient-specific full-length antibody library using mammalian cell surface display technique.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) were isolated from patients with renal cell carcinoma. The repertoires of kappa light chain (LCkappa) and heavy chain variable region (VH) of antibody were amplified by RT-PCR. The LCkappa and VH libraries were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO10 to construct the renal cell carcinoma patient-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length human antibodies expressed on the surface of 293T cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>The libraries of renal cell carcinoma-specific antibody kappa light chain (LCkappa) and heavy chain (IgG1) were constructed. The expression of the full-length human antibodies on the surface of 293T cell was confirmed by flow cytometry. The libraries showed an expressible combinatory diversity of 7.5x10(10).</p><p><b>CONCLUSION</b>The expressible antibody library provides a useful platform for screening of renal cell carcinoma-specific antibodies.</p>


Subject(s)
Amino Acid Sequence , Antibodies, Neoplasm , Allergy and Immunology , Antibody Specificity , Carcinoma, Renal Cell , Allergy and Immunology , Humans , Kidney Neoplasms , Allergy and Immunology , Molecular Sequence Data , Peptide Library
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