Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP / 生物工程学报

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.

Expression, purification, and characterization of fusion protein TAT-cytoglobin / 生物工程学报

he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.

HIV-1 Tat Protein Promotes Amyloid beta Generation and Tau Phosphorylation in Rat Hippocampal Slices

HIV-1 Tat protein has been implicated as a causative agent in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND) and Alzheimer's disease (AD)-like pathology in HIV-1 infected patients. Here, we provide insights into the potential roles of extracellular HIV-1 Tat protein in amyloid beta (Abeta) generation and Tau phosphorylation, two major neuropathological features of AD. Exposure of the rat hippocampal slices to the full-length HIV-1 Tat protein (Tat1-86) for 3 days led to the increased levels of Abeta precursor protein (APP) accumulation, which accompanied by Abeta generation in the hippocampus, the brain region most commonly damaged in HIV-1-associated dementia (HAD). Moreover, extracellular HIV-1 Tat significantly stimulated the level of phosphrylated Tau (pTau) identified using immunoblotting with AT8 antibody, which recognizes abnormally hyperphosphorylated Tau. Collectively, our data suggest that HIV-1 Tat plays important roles in increasing the levels of APP accumulation, Abeta generation and Tau phosphorylation in the hippocampus, and thereby might contribute to the development of AD-like pathology in HIV-1-infected patients.

HIV-1 Tat Protein-dependent Cytotoxicity is Attenated by 15-deoxy-Delta12,14-Prostaglandin J2 in Rat Hippocampal Slices: Involvement of the ERK1/2 Signaling Pathway

15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) may hold promise in treatment of the pathologies associated with human immunodeficiency virus (HIV) infection of the central nervous system. However, its precise role and neuroprotective mechanism in the hippocampus remain poorly understood. In the present study, rat hippocampal slices were stimulated with HIV-1 Tat protein to investigate the protective role of 15d-PGJ2 on the hippocampal cytotoxicity. Full-length HIV-1 Tat protein (Tat1-86), but neither its Tat32-62 nor Tat30-86 fragment, significantly induced cytotoxicity in the hippocampus, the brain region most commonly damaged in HIV-associated dementia. This Tat-induced cytotoxicity was associated with inactivation of MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, Tat1-86 did not alter Wnt signaling pathway necessary for cell survival. Pretreatment of slices with 15d-PGJ2 markedly reduced Tat-driven cytotxicity. Interestingly, this reduction was accompanied by suppression of ERK inactivation in response to Tat. Moreover, the inhibition of the MEK/ERK pathway with SL327 enhanced the Tat-induced cytotoxicity, confirming the ERK-dependent mechanism of Tat-driven cytotoxicity. Collectively, these data demonstrate that the protective action of 15d-PGJ2 against the hippocampal cytotoxicity upon Tat stimulation is exerted through suppression of Tat-mediated ERK1/2 inactivation.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Preparation and in vitro evaluation of pH-sensitive TAT peptide conjugated micelles / 药学学报

Doxorubicin loaded micelles were prepared by film-hydration method using stearyl sulfadiazine (SA-SD) which is pH sensitive, methoxy (polyethylene glycol)-2000-1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (mPEG-DOPE) and transactivator of transcription (TAT) peptide conjugated PEG-DOPE. Mean diameter of the pH-sensitive micelles was about 20 nm with a (99.1 +/- 2.1) % drug entrapment efficiency at pH 7.4. Flow cytometry studies revealed that the simple TAT micelles was taken up rapidly at the same level at pH 6.8 and pH 7.4. However, the pH-sensitive micelles entered the tumor cell less at pH 7.4 and significantly increase at pH 6.8. After 1 h incubation at pH 6.8, the amount of the pH-sensitive micelles taken up by cancer cell 4T1 was almost similar to simple TAT micelles. The confocal microscopy indicated that the pH-sensitive micelles entered the 4T1 cells at pH 6.8 more than at pH 7.4. It was indicated that the pH-sensitive micelles could shield TAT peptide at normal pH 7.4 and deshield it at pH 6.8. Hence, TAT peptides lead the drug-loaded micelles into the tumor cells and killed them selectively. The pH-sensitive micelle may provide a novel strategy for design of cancer targeting drug delivery system.

Construction, expression and immunogenicity analysis of a Tat N-terminus-deleted mutant fusion protein of human immunodeficiency virus type 1 / 病毒学报

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.

Pharmacokinetics of a fusion protein for human acidic fibroblast growth factor and transcriptional activator protein in rat and its penetration across blood-brain barrier / 药学学报

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

Tat transduction peptide displayed on the nucleocapsid improved the baculovirus transduction of mammalian cells / 生物工程学报

In order to improve the transduction efficiency of insect baculovirus in mammalian cells, we constructed two recombinant baculoviruses, AcRed-tat and AcRed. Both viruses expressed red fluorescence protein gene (dsRed) as a reporter in mammalian cell lines. AcRed-tat also contained the coding sequence of HIV-1 Tat transduction peptide fused with viral major capsid protein gene vp39 and enhanced green fluorescence gene (egfp) driven by virus polyhedrin promoter. It expressed the Tat fusion protein in infected insect cells, which was incorporated into the nucleocapsids of progeny virus. As a control, AcRed had the fusion gene of vp39 and egfp driven by polyhedrin promoter. Flow cytometry analysis demonstrated that although similar level of red fluorescence was produced in HEK23 cells transduced by the two recombinant viruses, significantly higher red fluorescence level was seen in CHO and Vero cells transduced by AcRed-tat than that by AcRed. These results suggested that Tat transduction peptide might improve the baculovirus-mediated gene expression in some mammalian cells. Our work provided a new approach to improve baculovirus as a gene delivery vector for mammalian cells.

CTL responses to regulatory proteins Tat and Rev in HIV-1 B'/C virus-infected individuals / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C virus-infected ART-naive individuals.</p><p><b>METHODS</b>HIV-1-specific CTL responses were analyzed by IFN-gamma ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05.</p><p><b>RESULTS</b>Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study.</p><p><b>CONCLUSION</b>Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.</p>

Comparison of two delivery system efficiency by using polyethylenimine [PEI] for plasmid HPV16E7 DNA transfection into COS-7 cells

DNA vaccines have been widely used to develop immunity against various pathogens including parasites and viruses. The potential of DNA vaccine to induce an effective immune response is related to the expression levels of the encoded protein in eukaryotic cells. Therefore, optimization of plasmid DNA delivery system is a major concern in protein expression in order to make an efficient DNA vaccination. Non-viral vectors such as polymers and cationic peptides have been recently known as efficient gene delivery systems into eukaryotic cells. In this study, transfection efficiency of HPV16E7 gene was evaluated by two non-viral delivery systems in vitro. DNA construct encoding HPV16E7 [pEGFP-E7] was prepared in large scale with high purity. Then, two delivery systems including polymer PEI 25 kDa and polymer-peptide hybrid as PEI600-Tat conjugate were used to compare their efficiency for HPV16E7 DNA transfection in vitro. Our data demonstrated that both delivery systems including PEI 25 kDa and PEI600-Tat in vitro, but its toxicity was obstacle in vivo. Therefore, with regard to low toxicity of PEI600-Tat delivery system and its potent plasmid DNA delivery, it is critical issue to study its potency as new delivery system in vivo

Extracellular HIV-1 Tat enhances monocyte adhesion by up-regulation of ICAM-1 and VCAM-1 gene expression via ROS-dependent NF-kappaB activation in astrocytes

One of characteristic features of AIDS-related encephalitis and dementia is the infiltration of monocytes into the CNS. HIV-1 Tat was demonstrated to facilitate monocyte entry into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of adhesion molecules, generation of reactive oxygen species (ROS) and NF-kappaB activation in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein and mRNA levels of ICAM-1 and VCAM-1, as measured by Western blot analysis and RT-PCR, indicating that Tat increases these protein levels at an mRNA level. In addition, Tat induced the activation of NF-kappaB in astrocytes. Treatment of CRT-MG with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of ICAM-1 and VCAM-1. Furthermore, HIV-1 Tat protein increased ROS generation. Inhibition of Tat-induced ROS generation by N-acetyl cysteine, vitamin C and diphenyl iodonium suppressed Tat-induced NF-kappaB activation, ICAM-1 and VCAM-1 expression, and monocyte adhesion in CRT-MG. These data indicate that HIV-1 Tat can modulate monocyte adhesiveness by increasing expression of adhesion molecules such as ICAM-1 and VCAM-1 via ROS- and NF-kappaB-dependent mechanisms in astrocytes.

Suppression of HIV-1 Tat-induced monocyte adhesiveness by a cell-permeable superoxide dismutase in astrocytes

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.

Bone regenerative effects of recombinant human bone morphogenetic protein-2 employed protein transduction domain / 대한치주과학회지

Bone morphogenetic proteins(BMPs) are regarded as members of the transforming growth factor-beta superfamily with characteristic features in their amino acid sequences. A number of studies have demonstrated the biologic activities of BMPs, which include the induction of cartilage and bone formation. Recently there was a attempt to overcome a limitation of mass production, and economical efficieny of rh-BMPs. The method producing PTD by using bacteria have advantages of acquiry a mass of proteins. Hences, a new treatment which deliver protein employed by protein transduction domain(PTD) has been tried. The purpose of this study was to evaluate the bone regenerative effect of TATBMP-2 and TAT-HA2-BMP-2 employed by PTD from HIV-1 TAT protein for protein translocation in the rat calvarial model. An 8mm calvarial, critical size osteotomy defect was created in each of 32 male Spraque-Dawley rats(weight 250~300g). The animals were divided into 4 groups of 32 animals each (4 animals/group/healing interval). The defect was treated with TATBMP-2/ACS(Absorbable collagen sponge) (TATBMP-2 0.1mg/ml), TAT-HA2-BMP-2/ACS(TAT-HA2-BMP-2 0.1mg/ml), ACS alone or left untreated for surgical control(negative control). The rats were sacrificed at 2 or 8 weeks postsurgery, and the results were evaluated histologically. The results were as follows: New bone formation were not significantly greater in the TATBMP-2/ACS group relative to negative, and positive control groups. New bone was evident at the defect sites in TAT-HA2-BMP-2/ACS group relative to negative, positive control and TATBMP-2 groups. There were a little bone regeneration in TATBMP-2 groups. While, enhanced local bone formation were observed in TAT-HA2-BMP-2 group. But, The results was not the same in all rat defects. Therefore, further investigations are required to develop a method, which disperse homogenously, and adhere to target cells.

Labeling Efficacy of Superparamagnetic Iron Oxide Nanoparticles to Human Neural Stem Cells: Comparison of Ferumoxides, Monocrystalline Iron Oxide, Cross-linked Iron Oxide (CLIO)-NH2 and tat-CLIO

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH2 and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5x105 HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microgram/ml of ferumoxides, MION or CLIO-NH2, and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH2, respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH2 into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH2 and the transfection agent PLL.

Cell-penetrating peptide-based functional study of p38 MAPK / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells.</p><p><b>METHODS</b>Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E. coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol.</p><p><b>RESULTS</b>The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS-PAGE, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manner. His-TAT-p38 was found capable of increasing the activity of endogenous p38 in ECV304 cells, but His-TAT-p38(AF) inhibited the phosphorylation of ATF2 so as to block the transduction of p38 signal pathway when the cells were stimulated with sorbitol.</p><p><b>CONCLUSION</b>p38 MAPK protein delivery system based on TAT protein has been constructed successfully. It is confirmed that TAT can transfer the proteins into the cells in a time- and dose-depended manner. TAT-p38 and its dominant negative form possess high biological activity after transduction into ECV304 cells by TAT protein delivery system, and the former can increase the activity of endogenous ATF2, but the latter inhibits the transduction of endogeneious p38 signal pathway in ECV304 cells with high osmotic stress.</p>

Effect of sulfated polymannuroguluronate on Tat induced proinflammatory cytokines release in THP-1 cells and its mechanism of action / 药学学报

<p><b>AIM</b>To investigate the effects of sulfated polymannuroguluronate (SPMG), a novel candidate anti-AIDS drug in Phase II clinical trial, on Tat-induced release of proinflammatory cytokines (i.e., TNFalpha, IL-1beta and IL-6) and its related mechanism.</p><p><b>METHODS</b>The effects of SPMG on Tat induced TNFalpha (4 h), IL-1beta and IL-6 (6 h) secretion in THP-1 cells were measured by ELISA. Western blotting analysis was used to study the effects of SPMG on Tat induced PKCzeta, PKCtheta and PKCsigma phosphorylation.</p><p><b>RESULTS</b>SPMG (50 to 100 microg x mL(-1)) markedly suppressed TNFalpha, IL-1beta and IL-6 secretion in Tat activated THP-1 cells. In THP-1 cells the phosphorylation levels of PKCzeta, PKCtheta and PKCsigma significantly increased following Tat stimulation, and only PKCsigma phosphorylation levels was inhibited by SPMG (50 to 100 microg x mL(-1)).</p><p><b>CONCLUSION</b>SPMG suppresses the secretion of proinflammatory cytokines in THP-1 cells may be by inhibiting PKCsigma activation.</p>

Extracellular HIV Tat and Tat cysteine rich peptide increase CCR5 expression in monocytes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In our previous work we reported that HIV Tat and 6 cysteine rich peptides of Tat induce tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in human monocytes (Yang et al., 2003). Here our results showed that HIV Tat and Tat cysteine rich peptide increase CCR5 expression in human monocytes, and this activity is inhibited by rabbit anti-Tat. Boiled Tat does not increase CCR5 expression in monocytes. These results provide insight into a new mechanism by which HIV Tat plays a key role in the pathogenesis of HIV-1 infection.

HIV TAT variants differentially influence the production of glucocerebrosidase in Sf9 cells

Gaucher disease, the most common lysosomal storage disorder, is currently treated with enzyme replacement therapy. This approach, however, is ineffective in altering the progression of neurodegeneration in type 2 and type 3 patients due to the difficulty of transferring the recombinant enzyme across the blood-brain barrier. Human immunodeficiency virus type 1 trans-activating transcriptional activator protein (HIV TAT) contains a protein transduction domain that can be added to a fusion protein partner to allow for transport of the partner across membranes. Consequently, we examined the creation, production, and secretion of fusion constructs containing glucocerebrosidase and either wild-type TAT or modified TAT in Sf9 cells. All three constructs exhibited successful expression, with wild-type TAT chimeras showing lower levels of expression than modified TAT chimeras.

Effect of HIV Tat protein on CCR5 expression in monocytes and infection with monocyte-tropic HIV strains / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the effects of HIV Tat protein on CCR5 expression of monocytes and HIV infection in monocytes.</p><p><b>METHODS</b>Membrane expression of CCR5 on monocytes was analyzed by flow cytometry. Stimulated with HIV Tat protein, monocytes were infected with monocyte-tropic HIV(Ba-L) and HIV gag p24 level in the supernatant was measured by ELISA methods.</p><p><b>RESULTS</b>HIV Tat protein increased CCR5 expression in human monocytes,which was inhibited by rabbit anti-Tat polyclonal antibody. Tat protein also increased p24 level after monocyte-tropic HIV-1(Ba-L) infected monocytes.</p><p><b>CONCLUSION</b>Tat increases CCR5 expression and HIV-1 infection in monocytes, which indicates that HIV Tat might be a key protein in HIV-1 infection.</p>