Wholesale MAC Cosmetics HIV  1 Tat protein bindi

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HIV  1 Tat protein binding Tar HIV medicine screening model of

Author: Lee to stay Liuchao Qi, Kun Zou, Feng-Lan Li, Han Yu, Fan Yang , Wanglei Li
Abstract purpose of the establishment of HIV-1 Tat Tar protein binding of anti HIV-1 drug screening model for anti-HIV drug screening. Methods Construction of HIV-1 Tat protein expression of the eukaryotic expression vector (pCDNA3.1 (+)-Tat) and HIV-1 LTR-luc luciferase reporter gene, using lipofectamine method of transfection of HeLa cells, fluorescence Tat protein was detected in promoting luciferase expression in HeLa cells, protein binding to establish HIV-1Tat Tar cell model for anti-HIV-1 drug screening. By DRB (5,6 - dichloro - 1 - β-furosemide nuclear nitrosourea - benzimidazole) as a positive control, to model and optimize the conditions. Results of repeated experiments show that the gene and the ratio of reporter gene and transfected into the culture medium when the calf serum concentration, cell density, transfection, the cell state and the length of such drugs on the model of stability and sensitivity of impact. Conclusion optimized cell model Bunge, basil extracts such as screening and analysis of the results.
Key words HIV-1 Tat LTR-Luc luciferase Jiyinyizi drug screening
Abstract: ObjectiveTo establish HIV-1 Tal binding to Tar model. MethodsHIV-1 Tat protein eukaryotic expression plasmid (pCDNA3.1 (+)-Tat) and luciferase report gene HIV-1 LTR-luc (LTR-LUC) were constructed and transfected into HeLa cells with lipofectamine 2000. The expression of HIV-1 Tat protein in HeLa cells was detected with Iuminometer. HIV-1 Tat protein binding tar model for screening HIV-1 inhibitors was thereby constructed.Results The abstractions of Xanthoceras sorbifolia and Perilla frutescens and so on were detected and DRB (5,6-dichloro-1-β-D-robofuranosylbenzimidazole) was positive control. The stability of the model was tested and regulated. ConclusionThe repeated experiments indicate that the matching of purpose gene and report gene, the rate of new calf serum in medium during transfection, density of the transfected cells, condition of the untransfected cells and the action time of drugs affect the stability and sensitivity of the model.
Key words: HIV-1 Tat; LTR-Luc luciferase gene; Model for screening
Human immunodeficiency virus (HIV) is the acquired immunodeficiency syndrome (AIDS) in the pathogen; is a reverse transcriptase RNA virus, known as the \curative drugs. Has approved the listing of drugs (HIV protease, reverse transcriptase inhibitors) can only ease the pain of expensive, toxic side effects,[link widoczny dla zalogowanych], easy to produce virus variability,[link widoczny dla zalogowanych], drug resistance, poor efficacy, offset, etc. [1,2]. Therefore, from the AIDS virus, basic research is not easy to find new targets of drug resistance become the focus. Of anti-HIV drugs currently focused on reverse transcriptase, protease, enzyme and virus adsorption integration, integration, and several other targets. HIV trans-activation factor (transactivator, Tat) as the HIV regulatory protein, not only through the regulation of trans-activating effect of viral gene replication and expression, also induced apoptosis, activation of quiescent T lymphocytes, suppression of the immune cell differentiation and proliferation, help spread the virus infection and the body, leading to various complications [3,4]. Tat and trans-activation effect of area (trans-activator responseregion, TAR) RNA interaction, trans-activation of transcription. Tat and TAR structure are highly conserved, making it the search for new mechanisms and not easy to produce drug resistance AIDS drugs for a very attractive new target [5].
Tat gene encodes proteins with HIV-1 virus long terminal repeat (LTR) combine to increase viral gene transcription rates for all,[link widoczny dla zalogowanych], but also in the transcriptional promoting translation of viral mRNA, resulting in increased virus replication. [6] In this study, through the establishment of HIV-1 Tat and LTR binding, resulting in luciferase reporter gene was highly expressed in cells, Tat, and LTR sequences reflect the effective integration, the establishment of Tat trans-activation effect of regulation of viral gene replication and expression of model so for drugs active ingredient of natural selection, which is simple, fast, trace, low cost, safe, free from restrictions on P3 laboratory, without isotopic features for the establishment of anti-HIV-1 laboratory drug screening model to provide reference and guidance, are of great practical significance.
1 Material and Methods
1.1 Materials
HIV-1 Tat protein in the eukaryotic expression plasmid pCDNA3.1 (+)-Tat, and reporter gene HIV-1 LTR-luc (LTR-LUC) by the Construction of Three Gorges University, Institute of Molecular Biology save. HeLa cell line from Beijing Union Medical College favors. Lipofectamine 2000, Opti-MEM medium are Invitrogen Corporation reagents, newborn calf serum (NCS) for the GIBCO products. DMEM medium, trypsin, substrate Luciferin, positive control DRB, cell lysate 1,2-diaminocylclohexane-N, N, N, N-tetraacetic acid, Coomassie brilliant blue and other medicines are Sigma products.
1.2 Methods
Constructed HIV-1 Tat protein expression of the eukaryotic expression vector (pCDNA3.1 (+)-Tat), HIV-1 LTR-luc luciferase reporter gene, using lipofectamine method HeLa cells were transfected fluorescence was detected in HeLa cells Tat protein expression, protein binding establishment of HIV-1Tat Tar anti-HIV-1 drug screening. Transfected cells added to the cell toxicity of the drug in 20% concentration, to DRB as a positive control (Tat / Tar inhibitor, Mancebo et al., 1997; Nekhai et al, 1997), After the culture collection of cell lysis supernatant of the relative fluorescence value (RLU), Coomassie brilliant blue (G-250) Determination of protein content, protein content in the uniform conditions, drugs inhibit the expression of Tat protein expression for drug group and the difference between the control group RLU value.
1.2.1 HeLa cells by liposome transfection experiments Lipo fectamine 2000's manual to operate: ① 1 day before transfection HeLa cells inoculated with 2 × 105cells/ml concentration to 24 well plates, 0.5 ml / hole Purchase 37 ℃, 5% CO2 incubator overnight. ② transfection the transfected cells pre-treated for 1h equal volume, fresh DMEM medium without antibiotics, home 37 ℃, 5% CO2 in an incubator incubated 1h. ③ DNA-Lipofectamine precipitates Preparation: A: pCDNA3.1 (+)-Tat and LTR-Luc diluted and mixed: pCDNA3.1 (+)-Tat and LTR-Luc concentration was 0.1 μg / μl, the two diluted into a certain proportion of the common medium Opti-MEM. B: Lipofectamine diluted: with Opti-MEM at room temperature after dilution required 5 min. C: the preparation of good A liquid and B liquid mixture, beating slowly mixing, at room temperature for 20 min. D: to C by adding liquid slowly to be transfected HeLa cells, mixing, incubation 4 ~ 6 h. E: discard the supernatant, the complete change of fresh DMEM medium, incubated for 16 ~ 48 h. F: Results test: discard the supernatant, adding a certain amount of cell lysate, cell supernatant collected the corresponding fluorescent enzyme substrate, fluorescence was detected RLU, and Coomassie brilliant blue (G-250) Determination of protein content.
1.2.2 HIV-1 Tat protein binding Tar anti-HIV-1 drug screening Model experiments established by the above methods,[link widoczny dla zalogowanych], for drug screening. Specific methods: \, and the supernatant 10 μl substrate luciferin100 μl,[link widoczny dla zalogowanych], Sirius Berthold luminemeter fluorescence analyzer test results.
1.2.3 HIV-1 Tat protein binding of anti-HIV-1 Tar screening model for the stability of optimal experimental model through experiments found less stable, drug inhibition was not significant. To this end, a number of factors the model debugging. Specific methods: DRB to the positive control, negative control with blank group to sorbifolia kinds of skin, basil extracts of soluble material was tested drug, the establishment of 3 holes, 3 repeated experiments, by changing the purpose of the ratio of gene and reporter gene and transfected into the culture medium when the calf serum concentration, the number of the transfected cells were inoculated (after determining the ratio of two recombinant plasmids using cell culture bottles with 3 × 105 cells / ml, passage, 6 ml / bottle), drug content, before the cells transfected with the state and the duration of drug action such factors as stability of the model.
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