RESUMEN
<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>
Asunto(s)
Animales , Humanos , Secuencia de Aminoácidos , Anticuerpos , Genética , Alergia e Inmunología , Especificidad de Anticuerpos , Artritis Reumatoide , Alergia e Inmunología , Técnicas de Visualización de Superficie Celular , Clonación Molecular , Escherichia coli , Genética , Metabolismo , Vectores Genéticos , Genética , Células HEK293 , Inmunoglobulina G , Genética , Cadenas Pesadas de Inmunoglobulina , Genética , Cadenas kappa de Inmunoglobulina , Genética , Linfocitos , Alergia e Inmunología , Metabolismo , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes , Genética , Alergia e Inmunología , TransfecciónRESUMEN
<p><b>OBJECTIVE</b>To establish immortalized B lymphoblast cell lines (B-LCLs) from healthy anti-HBs antibody (anti-HBs)-positive volunteers and screen for human anti-HBs and the antibody-secreting cells.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMC) isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus (EBV) and incubated in the presence of CpG DNA motifs and cyclosporin A (CyA). The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs.</p><p><b>RESULTS</b>Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138.</p><p><b>CONCLUSION</b>Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.</p>