ABSTRACT
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.
Subject(s)
Humans , Cell Line , Down-Regulation , Genetic Therapy , Genetic Vectors , Genetics , Metabolism , HIV Infections , Therapeutics , Virology , HIV-1 , Genetics , Metabolism , Lentivirus , Genetics , Metabolism , RNA Interference , RNA, Small Interfering , Genetics , Metabolism , Therapeutic Uses , tat Gene Products, Human Immunodeficiency Virus , Genetics , Metabolism , vpr Gene Products, Human Immunodeficiency Virus , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the effect of anti-HIV III B virus with extractions from Juglans regia, so as to searching for the new and efficacious leading compound of AIDS therapy.</p><p><b>METHOD</b>Phytochemical and chromatographical techniques were used to isolate compounds from J. regia; MT4 cells and HIV III B virus were used to study the effect of anti-HIV activity in vitro. BIACORE 3000 molecule coupled equipment were used for the target research also.</p><p><b>RESULT</b>Two extractions (B&E) were isolated from J. regia which possess the effect of anti-HIV activity. Targets study found that extraction B could affected on HIV-1 gp-41 fusing protein and extraction E could affected on HIV-1 integrase respectively.</p><p><b>CONCLUSION</b>J. regia is a traditional Chinese medicine with active anti-HIV activity and worth to be studied further.</p>
Subject(s)
Humans , Cell Line , Drugs, Chinese Herbal , Chemistry , Pharmacology , HIV-1 , Juglans , Chemistry , Plant Extracts , Chemistry , PharmacologyABSTRACT
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) cDNA was amplified from total RNA prepared from nonpermissive H9 cells by RT-PCR. APOBEC3G cDNA is 1155nt long, encoding 384 amino acids. The APOBEC3G gene was then cloned into the eukaryotic expression vector pEGFP-C3. The generated pEGFP-3G construct was then transfected into CD4+ HeLa cell to determine the expression and the subcellular localization of GFP-APOBEC3G fusion protein. Under CLSM the localization of the expressed GFP-APOBEC3G in the cytoplasm of CD4+ HeLa cells was observed.
Subject(s)
Humans , APOBEC-3G Deaminase , Cell Line , Cytidine Deaminase , Cytoplasm , Metabolism , DNA, Complementary , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Metabolism , HeLa Cells , Microscopy, Confocal , Methods , Nucleoside Deaminases , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Repressor Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To probe into the feasibility of screening anti-HIV compounds by using HIV-1 p24 detection kit made by Hebei Medical University.</p><p><b>METHODS</b>The sensitivity, reproducibility and efficacy of the Hebei p24 kit were evaluated compared with the commercially available Vironostika HIV-1 Antigen Microelisa System (Biomerieux).</p><p><b>RESULTS</b>Hebei p24 kit had high sensitivity and good reproducibility. In vitro screening demonstrated that there was no statistically significant difference (P greater than 0.05) between these two kits in assessing anti-HIV compounds.</p><p><b>CONCLUSION</b>Hebei p24 kit could be used as an easily affordable alternative method for detection of HIV-1 in screening anti-HIV compounds.</p>
Subject(s)
Humans , Anti-HIV Agents , Pharmacology , Cell Line , Drug Evaluation, Preclinical , Methods , Feasibility Studies , HIV Core Protein p24 , HIV-1 , Allergy and Immunology , Reagent Kits, Diagnostic , Reference Standards , Reproducibility of ResultsABSTRACT
This paper summarized the recent 6 years' progress of anti-HIV compounds and traditional Chinese medicines by searching international network and reviewing the domestic and foreign literature. Traditional Chinese medicinal appeared to be a rich source of potentially useful materials for the treatment of human immunodeficiency virus infection. Some of them are much more potent in anti-HIV activity. And some components extracted from the herbs are even more tonic than the crude herb medicines. It has been proved that some active components such as alkaloids, proteins, flavonoids, quercetin, terpene, lignanoid are able to work on anti-HIV. People should pay more attention to the study of traditional Chinese medicine and the leading compounds on anti-HIV/AIDS in the clinic and in the laboratory. So searching for high efficacy and low toxicity anti-HIV drug from traditional Chinese medicine is an important and prospective research direction in the future.
Subject(s)
Animals , Humans , Acquired Immunodeficiency Syndrome , Drug Therapy , Adjuvants, Immunologic , Pharmacology , Anti-HIV Agents , Pharmacology , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , HIV , Medicine, Chinese Traditional , Phytotherapy , Plants, Medicinal , ChemistryABSTRACT
<p><b>OBJECTIVE</b>Chimeric human/bovine immunodeficiency virus (HBIV) cDNA was constructed by replacing HIV tat and LTR with bovine immunodeficiency virus (BIV) tat and LTR to study the activity of BIV tat and LTR in the chimerae.</p><p><b>METHODS</b>The target fragments of BIV tat, LTR and HIV gag, pol, env were respectively amplified by using PCR and sequentially inserted into pBluescript SK(+) vector. The chimeric clone was transfected into human MT4 cells. The transcript and gene expression of the HBIV chimeric virus were detected by using RT-PCR and a reverse transcriptase assay, respectively.</p><p><b>RESULTS</b>BIV tat mRNA and HIV gag mRNA were detected. The reverse transcriptase activity of the chimeric virus was analyzed in the fluctuation curve.</p><p><b>CONCLUSIONS</b>In chimeric HBIV cDNA transfected MT?4 cells, BIV tat and HIV gag were transcripted. The reverse transcriptase of the chimeric virus had biological activity. These data suggest that in MT4 cells, BIV LTR had promoter activity and BIV tat had the function of transactivation in the chimeric virus. The study of the chimeric virus with infectivity is in progress.</p>