RESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a primary liver malignancy that mainly occurs in patients with chronic liver disease and cirrhosis. Risk factors for HCC include hepatitis B virus (HBV) infection. However, the specific role of HBV infection in HCC development is not yet completely understood. In order to reveal the effects of HBV on HCC, we compare the genes of HCC patients infected with HBV with those who are not infected. METHODS: We encoded the genes of these two types of HCC in databases using enrichment scores of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway terms. A random forest algorithm was employed in order to distinguish these two types in the classifier, and a series of feature selection approaches was used in order to select their optimal features. Novel HBV-associated and -non-associated HCC genes were predicted, respectively, based on their optimal features in the classifier. A shortest-path algorithm was also employed in order to find all of the shortest-paths genes connecting the known related genes. RESULTS: A total of 54 different features between HBV-associated and -non-associated HCC genes were identified. In total, 1236 and 881 novel related genes were predicted for HBV-associated and -non-associated HCC, respectively. By integrating the predicted genes and shortest path genes in their gene interaction network, we identified 679 common genes involved in the two types of HCC. CONCLUSION: We identified the significantly different genetic features between two types of HCC. We also predicted related genes for the two types based on their specific features. Finally, we determined the common genes and features that were involved in both of these two types of HCC.
Assuntos
Biomarcadores/análise , Carcinoma Hepatocelular/genética , Vírus da Hepatite B/genética , Hepatite B/complicações , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Estudos de Casos e Controles , Hepatite B/genética , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.
Assuntos
Biblioteca Gênica , Anticorpos Anti-Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Afinidade de Anticorpos , Células CHO , Clonagem Molecular , Cricetinae , Epitopos/imunologia , Hepatite B/prevenção & controle , Hepatite B/transmissão , Anticorpos Anti-Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Humanos , Ligação Proteica , Vacinas contra Hepatite Viral/genéticaRESUMO
OBJECTIVE: To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries. METHODS: 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. RESULTS: 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). CONCLUSION: The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.
Assuntos
Anticorpos/imunologia , Artrite Reumatoide/imunologia , Técnicas de Visualização da Superfície Celular , Imunoglobulina G/biossíntese , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos/genética , Especificidade de Anticorpos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Células HEK293 , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , TransfecçãoRESUMO
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.
