Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.
Cell Line ; Down-Regulation ; Genetic Therapy ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; therapy ; virology ; HIV-1 ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; therapeutic use ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism ; vpr Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

Apoptosis ; Hepatocytes ; pathology ; Humans ; Trans-Activators ; physiology

OBJECTIVETo highly express TAT-HBX-EGFP fusion protein and study its distribution in mouse liver.

METHODSTAT-HBX-EGFP recombinant vector was constructed and fusion protein was induced by IPTG and expression in BL21; fusion protein was purified by Ni-NTA argarose, then injected into the peritoneal cavity of the mice. Distribution of fusion protein was observed by immunofluorescence.

RESULTSTAT-HBX-EGFP was highly expression in E. coli; HBX could be induced into mouse liver by TAT.

CONCLUSIONHBX protein could be induced into mouse liver by TAT induced peptide.

Animals ; Cell Membrane ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Green Fluorescent Proteins ; genetics ; isolation & purification ; metabolism ; Hepatitis B ; metabolism ; virology ; Humans ; Liver ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Protein Transport ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Trans-Activators ; genetics ; isolation & purification ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; isolation & purification ; metabolism ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; isolation & purification ; metabolism

BACKGROUND: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and transactivator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. METHODS: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-gamma producing T lymphocytes were measured. RESULTS: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-gamma secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+ T cell to CD4+ T cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. CONCLUSION: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.
Carcinoembryonic Antigen ; Cytoplasm ; Dendritic Cells ; Epithelial Cells ; Humans ; Immunization ; Immunotherapy ; Lymphocytes ; T-Lymphocytes ; Trans-Activators

This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli.
Escherichia coli* ; Escherichia* ; HIV-1* ; Membranes ; Paraquat ; RNA, Messenger* ; Solubility ; Superoxide Dismutase ; Superoxides* ; Trans-Activators

Cell Differentiation ; Cell Line, Tumor ; metabolism ; Cell Proliferation ; Humans ; Trans-Activators ; biosynthesis ; genetics ; Transfection

Cardiovascular Diseases ; metabolism ; pathology ; Humans ; Smad Proteins ; metabolism ; Trans-Activators ; metabolism

As a protein originally found in plant pathogenic bacteria, transcription activator-like effectors (TALEs) can be fused with the cleaving domain of restriction endonuclease (For example Fok I) to form artificial nucleases named TALENs. These proteins are dependent on variable numbers of tandem Repeats of TALEs to recognize and bind DNA sequences. Each of these repeats consists of a set of approximately 34 amino acids, composed of about 32 conserved amino acids and 2 highly variable amino acids called repeat variant di-residues (RVDs). RVDs distinguish one TALE from another and can make TALEs have a simple cipher for the one-to-one recognition for proteins and DNA bases. Based on this, in theory, artificially constructed TALENs could recognize and break DNA sites specifically and arbitrarily to perform gene knockout, insertion or modification. We reviewed the development of this technology in multi-level and multi species, and its advantages and disadvantages compared with ZFNs and CRISPR/Cas technology. We also address its special advantages in industrial microbe breeding, vector construction, targeting precision, high efficiency of editing and biological safety.
Amino Acid Motifs ; Biotechnology ; DNA ; chemistry ; Endonucleases ; chemistry ; Tandem Repeat Sequences ; Trans-Activators ; chemistry

OBJECTIVESTo study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).

METHODSBoth 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.

RESULTSBoth 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.

CONCLUSIONSHBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Histone Deacetylase 1 ; metabolism ; Humans ; Hydroxamic Acids ; metabolism ; Immunoprecipitation ; Plasmids ; Protein Interaction Mapping ; Trans-Activators ; metabolism

OBJECTIVETo construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function.

METHODSThe mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast.

RESULTSThe subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function.

CONCLUSIONA subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

Cloning, Molecular ; Gene Library ; RNA, Messenger ; analysis ; Trans-Activators ; physiology ; Transcriptional Activation