ABSTRACT
<p><b>OBJECTIVES</b>To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).</p><p><b>METHODS</b>Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.</p><p><b>RESULTS</b>Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.</p><p><b>CONCLUSIONS</b>HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.</p>
Subject(s)
Humans , Histone Deacetylase 1 , Metabolism , Hydroxamic Acids , Metabolism , Immunoprecipitation , Plasmids , Protein Interaction Mapping , Trans-Activators , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the features of HBx protein distributed in liver cells and its expression in E. coli.</p><p><b>METHODS</b>The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography.</p><p><b>RESULTS</b>The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded.</p><p><b>CONCLUSION</b>This study may provide a basis for further study on the biological function of HBx at the protein level.</p>
Subject(s)
Humans , Carcinoma, Hepatocellular , Pathology , Cell Line , Cloning, Molecular , Escherichia coli , Metabolism , Genetic Vectors , Glutathione Transferase , Genetics , Hepatocytes , Cell Biology , Metabolism , Liver , Cell Biology , Liver Neoplasms , Pathology , Mutation , Recombinant Fusion Proteins , Genetics , Trans-Activators , Genetics , Tumor Cells, CulturedABSTRACT
The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.
ABSTRACT
A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Subject(s)
Bacteriophage lambda , Genetics , DNA, Recombinant , Genetics , Escherichia coli , Genetics , Genetic Engineering , Plasmids , Genetics , Rec A Recombinases , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene.</p><p><b>METHODS</b>Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression.</p><p><b>RESULTS</b>NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice.</p><p><b>CONCLUSION</b>Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.</p>
Subject(s)
Animals , Mice , Cell Line, Transformed , Cell Transformation, Neoplastic , Gene Expression , Genes, Neoplasm , Physiology , Mice, Inbred BALB C , Mice, Nude , NIH 3T3 Cells , Neoplasm Proteins , Genetics , Physiology , RNA, Messenger , Genetics , Random Allocation , TransfectionABSTRACT
Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.