RESUMO
Apoptin is a small molecular weight protein derived from chicken anemia virus, which can induce the apoptosis of transformed cells and tumor cells and leave primary and nontransformed cells unharmed. Apoptin's cell localization depends on its own phosphorylation state and cell type. In tumor cells, phosphorylated apoptin enters the nucleus and induces apoptosis. While, in normal cells apoptin mainly exists in the cytoplasm. Apoptin, as a disordered protein in cells, interacts with many proteins in cell signal pathways to induce apoptosis of tumor cells. The specific mechanism of apoptosis induced by apoptin has not been completely elucidated. Therefore, apoptin has become a potential anticancer agent. This review summarizes the research results of apoptin in our laboratory and reveals the specific antitumor mechanism of apoptin expressed by oncolytic virus vector on a variety of tumor cells and mouse models.
Assuntos
Antineoplásicos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Neoplasias/metabolismo , Vírus Oncolíticos/genética , Animais , Apoptose , Vírus da Anemia da Galinha/química , Humanos , Camundongos , Neoplasias/patologia , Neoplasias/terapia , FosforilaçãoRESUMO
UNLABELLED: Phylogenetic analysis of nucleotide sequences or deduced amino acid sequences of phosphoprotein (P protein), matrix (M) protein, and glycoprotein (G protein) genes of 18 Chinese isolates of Rabies virus (RABV) from 2003-2007 showed that these isolates formed a separate monophyletic lineage consisting of sub-lineages A and B. Compared with laboratory-fixed strains, recent Chinese isolates of sub-lineage B contained Val or Ala instead of Met69 in P protein, which is involved in generating truncated P proteins. In addition, one of these isolates CHpg3 had Pro instead of Ser63 and Leu instead of Ser64. Importantly, all functional domains of P and M proteins of all recent Chinese isolates were similar to those of laboratory-fixed strains. This study showed that although the recent Chinese RABV isolates belonged to a distinct lineage, their functional domains of P and M proteins were highly conserved. KEYWORDS: rabies virus; glycoprotein; phosphoprotein; matrix protein; China.
Assuntos
Genes Virais , Fosfoproteínas/genética , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Proteínas da Matriz Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , China , Primers do DNA/genética , DNA Viral/genética , Cães , Evolução Molecular , Chaperonas Moleculares , Dados de Sequência Molecular , Filogenia , Raiva/veterinária , Raiva/virologia , Vírus da Raiva/classificação , Homologia de Sequência de Aminoácidos , Suínos , Fatores de TempoRESUMO
The HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V(H)) and light (V(L)) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly(4)Ser)(3) to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 microg/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sequência de Bases , Relação Dose-Resposta Imunológica , Genes de Imunoglobulinas , Vetores Genéticos , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , HIV-1/fisiologia , Humanos , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais Cultivadas , Replicação Viral/imunologiaRESUMO
The recent H5N1 avian influenza outbreaks in Asia spread over more than 8 countries. It has caused enormous economic loss and grand challenges for the public health. During these breakouts we isolated three strains of H5N1 Avian Influenza Virus (AIV) from chickens and one from duck in different farms of Southern China. We completely sequenced these four AIVs. Molecular characterization demonstrated that these strains retain the reported H5N1 AIV sequence properties relevant to virus virulence and host adaptation. Phylogeny results demonstrated that three of these isolates (except A/Chicken/Guangdong/174/04) were closely linked to other H5N1 AIVs isolated from the recent H5N1 outbreaks in Asia. Six of 8 segments (except PA and M) of A/Chicken/Guangdong/174/04 also shares a close linkage to other H5N1 AIVs isolated from the recent H5N1 outbreaks. However, the PA gene of A/Chicken/Guangdong/174/04 and another H5N1 strain forms a distinct subgroup along with an H6N1 AIV, and the M gene of A/Chicken/Guangdong/174/04 shows a close linkage to some H5N1 AIVs from aquatic species in China. Our findings suggest that a new genotype of AIV (in addition to previous reported ones) was present during the 2003-04 Asian bird flu outbreaks and that continuing virus surveillance of AIVs be conducted to monitor the evolutionary paths of the A/Chicken/Guangdong/174/04-like AIVs.
Assuntos
Surtos de Doenças , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Animais , Galinhas , China/epidemiologia , Patos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Dados de Sequência Molecular , Filogenia , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNARESUMO
To achieve high-level expression of HIV-2(ROD) external glycoprotein gp105 in Pichia pastoris, the gp105 gene mutant tP1, with the 5' non-functional region of the gp105 gene removed, was obtained by PCR amplification and was cloned into secreted expression vector pHILS1. The His(+)Mut(s) recombinant P. pastoris strain was screened by PCR and induced by methanol. SDS/PAGE and Western-blot analyses showed that mutation of the low-usage codon AGG into synonymous codon CGA and the introduction of the optimal codon TTC made P. pastoris overexpress tP1, an 85 kDa heterologous glycoprotein that was secreted into the medium and recognized specifically by HIV-2 polyclonal antibody. The recombinant strain GS115/tP1 had excellent genetic stability in terms of the properties of growth and expression of gp105, and seven out of 58 recombinant stains with a yield of 29% were selected to be used for further purification of gp105.
Assuntos
Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , HIV-2/genética , Mutação , Pichia/metabolismo , Western Blotting , Clonagem Molecular , Códon , Eletroforese em Gel de Poliacrilamida , Produtos do Gene env/química , Glicoproteínas/química , Histidina/metabolismo , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
The biological and immunological characteristics of HIV-1 core protein p17-24 expressed by recombinant vaccinia viruses were studied. The results of indirect immunofluorescence assay (I-IFA), Western blot and dot ELISA showed that the two recombinants could express the p24 gag and p17-24 gag fusion proteins in infected cell lines respectively. The electromicroscope observation revealed that the expressed proteins could also assemble into virus-like particles. The recombinant vaccinia viruses can also stimulate mice for the formation of anti HIV-1 Gag p24 antibody. When infected with the recombinant viruses, the chromosome DNA ladder caused by the apoptosis of the BHK cell was observed.
Assuntos
Produtos do Gene gag/biossíntese , Antígenos HIV/biossíntese , Proteína do Núcleo p24 do HIV/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Virais , Animais , Anticorpos Anti-HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Camundongos , Vaccinia virus/genética , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
We devised vaccinia virus (VV)-based vector systems that support higher levels of expression of cloned genes in the early and late phases of the infection cycle than reported previously. Enhanced expression can be obtained by combining the promoter of the A-type inclusion body protein gene, the mutated early region of the 7.5-kDa gene promoter (7.5-kDa promoter), and the promoter of the hemagglutinin (HA) gene. One construct produced 60 micrograms/10(6) cells of chloramphenicol acetyltransferase (CAT), equivalent to 10-18% of total cell protein. Another construct produced about seven times more CAT during the early phase than the amount synthesized under the control of the 7.5-kDa promoter alone. The envelope proteins of human immunodeficiency virus type I synthesized during the early phase of infection were more active as immunogens than these proteins synthesized during the late phase, regardless of the amounts produced.
