ABSTRACT
Background: The human immunodeficiency virus type 1 [HIV-1] is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome [AIDS]. The aim of this study was to construct an RNA-positive control based on armored [AR] RNA technology, using HIV-1 RNA as a model
Methods: The MS2 maturase, a coat protein gene [at positions 1765 to 1787] and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 [DE3], and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside [IPTG] at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography
Results: The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33
Conclusions: Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory
ABSTRACT
Aim: Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs
Background: Gene therapy with adenovirus [Ad] vectors that express herpes simplex virus thymidine kinase [HSVtk] and ganciclovir [GCV] have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation
Methods: We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'- UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines
Results: Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively
Conclusion: Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression
ABSTRACT
Objective: To survey the level and patterns of reverse transcriptase-based drug resistance and subtype distribution among antiretroviral-treated HIV-infected patients receiving only reverse transcriptase inhibitors in Iran. Methods: A total of 25 samples of antiretroviral therapy experienced patients with no history of using protease inhibitors were collected. After RNA extraction, reverse transcriptase-nested PCR was performed. The final products were sequenced and then analysed for drug-resistant mutations and subtypes. Results: No drug resistant mutations were observed among the 25 subjects. The results showed the following subtypes among patients: CRF 35_AD (88%), CRF 28_BF (8%), and CRF 29_BF (4%). Conclusions: A significant increase in drug resistance has been noted in recently-infected patients worldwide. Subtype distributions are needed to perform properly-designed surveillance studies to continuously monitor rates and patterns of transmitted drug resistance and subtypes to help guide therapeutic approaches and limit transmission of these variants.
ABSTRACT
Background: Designing novel therapeutic agents has been a critical challenge for HIV disease
Materials and Methods: In current study a DNA sequence which was encoded the Tat protein was synthesized and inserted in pET 28 vector. Vector was cloned in BL21-DE3 E. coli and cultured in TB media. After protein expression, recombinant Tat protein was purified by NTA affinity chromatography. The Tat purified protein efficiency and confirmed by SDS-PAGE and Western blot, respectively. We were immunized the camel against HIV-1 Tat recombinant protein to made a camelid antibody library. Total RNA was extracted from camel lymphocytes and VHH fragments synthesized and amplified using RT-PCR and Nested- PCR methods by special primers
Results: The 350- 450 bp VHH gene fragment was produced by RT-PCR and Nested- PCR and extracted from agarose gel 1%. Then gel extraction was performed and pure fragments were inserted in HEN-4 vector by T4 DNA ligase
Conclusion: The library can be applied for biopanning and isolation of nanobody against HIV-1 Tat Protein. Nanobody small size may be a useful drug for treatment of HIV disease because give them the potency of the recognizing the cryptic epitopes of tat and neutralized the virus
ABSTRACT
Identification of drug resistant mutations is important in the management of HIV-1 infected patients. The aim of the current study was to evaluate drug resistance profile of RT gene and assess subtypes among the HIV-1 circulating strains and intensification of physician's options for the best therapy. HIV-1 RNA of 25 samples was extracted from plasma and RT Nested- PCR was performed and the final products were sequenced and phylogenetically analyzed. Stanford HIV drug resistance sequence database was used for interpretation of the data. The results of phylogenetic analysis showed subtypes A1 and B in 14 [58%] and 10 [42%] patients respectively. Of the 24 patients, 16 [66.6%] had resistance to NRTIs, 8 individuals [32%] to NNRTIs and one patient was susceptible to NRTIs as well as NNRTIs. The drug resistance interpretation in this study showed: 87.7% susceptible for AZT, 70.8% susceptible, and 25% high-level resistance for 3TC, 87.7% susceptible for TDF, 29.1% high-level resistance for NVP and 70.8% susceptible and 25% high-level resistance for EFV. Our data suggests that probably, the use of 2 NRTIs plus 1 protease inhibitor [PI] regimen is more effective than 2 NRTIs plus 1 NNRTI regimen in Iranian patients that use 2 NRTI plus NNRTI regimen and also continuous surveillance should be perform to evaluate resistance patterns for more effective therapeutic approaches