ABSTRACT
Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.
Subject(s)
Antiviral Agents , Pharmacology , Escherichia coli , Genetics , Genetic Engineering , Hepacivirus , Genetics , Physiology , RNA, Catalytic , Genetics , Pharmacology , Genetics , Ribonuclease P , Genetics , Viral Core Proteins , GeneticsABSTRACT
Measles and rubella virus cause fever rash diseases that are uneasy to differentiate clinically from each other. Specific primers and fluorescence-labeled probes were designed, and a multiplex Real-time RT-PCR with an internal control was developed to simultaneously identify the measles and rubella virus. The multiplex Real-time RT-PCR assay was specific and no false positive or false negative results were found. The sensitivity of the assay was 0.1TCID50/mL and 1TCID50/mL for measles and rubella virus respectively. Analysis with 0.1-10(3)TCID50/mL measles or rubella virus samples demonstrated high validity and reproducibility with the coefficient of variability(CV) of below 0.9% for both measles and rubella virus. Using ribonuclease P (RNase P) as internal false negative control, the developed multiplex Real-time RT-PCR assay is suitable for rapid clinical diagnosis of measles and rubella virus.
Subject(s)
Humans , Fluorescent Dyes , Measles , Diagnosis , Virology , Measles virus , Genetics , RNA, Viral , Genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Ribonuclease P , Genetics , Rubella , Diagnosis , Virology , Rubella virus , Genetics , Sensitivity and SpecificityABSTRACT
External Guide Sequences (EGSs) represents a novel nucleic acid based gene interference approach to modulate gene expression. They are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. DNA-based EGS1386 with a size of 12 nt was chemically synthesized to target the mRNA coding for the UL49 gene of human cytomegalovirus (HCMV). The DNA-based EGS1386 molecule efficiently directed human RNase P to cleave the target mRNA sequence in vitro. A reduction of more than 50% in the levels of UL49 expression was observed in human cells treated with the DNA-based EGS1386 targeted UL49 assayed by fluorescent quantization PCR and Western blotting. This results showed that the DNA-EGS1386 can effectively guide the RNase P cut the target mRNA. Therefore, DNA-EGS can develop into a new gene silencing technology and potential of the anti-viral reagents.
Subject(s)
Humans , Base Sequence , Cytomegalovirus , Genetics , Metabolism , Cytomegalovirus Infections , Virology , DNA, Viral , Genetics , Directed Molecular Evolution , Methods , Gene Expression Regulation, Viral , Nucleic Acid Conformation , Oligodeoxyribonucleotides , Genetics , Pharmacology , Chemistry , Pharmacology , RNA, Messenger , Genetics , Metabolism , Ribonuclease P , Genetics , Metabolism , Viral Structural Proteins , Genetics , MetabolismABSTRACT
This study was purposed to investigate the inhibitory effect of anti-C II TA M1-RNA on MHC II expression. The M1-RNA with guide sequences (GS) recognizing C II TA at 3408 site (M1-3408-GS) and C II TA target RNA (3176 - 3560) were constructed, then cloned into the pUC19 and pGEM-7zf (+) vector respectively. The recombinant M1-RNA and its target RNA were incubated in cell-free conditions. It showed that M1-3408-GS could exclusively cleave target RNA, then it was cloned into the psNAV vector. Stable transfectants of Jurkat cells with M1-3408-GS were analyzed for classical MHC II (HLA-DR, -DP, -DQ) induction in response to IFN-gamma by flow cytometry. The level of C II TA mRNA was measured by RT-PCR. The results showed that after IFN-gamma treatment, the expression of HLA-DR, HLA-DP, HLA-DQ on M1-3408-GS positive Jurkat cells decreased 83.17%, 94.12% and 84.31% respectively as compared with control. At the same time the mRNA contents of C II TA also markedly decreased (P < 0.05, t = 4.89). It is concluded that anti-C II TA M1-RNA (M1-3408-GS) inhibits C II TA, decreases itself mRNA content and so suppresses expression of MHC II molecules regulated by C II TA.
Subject(s)
Humans , Down-Regulation , Histocompatibility Antigens Class II , Metabolism , Jurkat Cells , Major Histocompatibility Complex , Genetics , Nuclear Proteins , Metabolism , RNA, Messenger , Metabolism , Ribonuclease P , Metabolism , Trans-Activators , MetabolismABSTRACT
An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of 1.4x10(7). This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.
Subject(s)
Catalytic Domain , Clone Cells , Gene Expression , Genomics , Repression, Psychology , Ribonuclease P , Ribonucleases , RNA , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To determine the effects of M1-GS RNA (M1 RNA) on bcr-abl mRNA and oncoprotein after M1 RNA with guide sequence (M1-GS RNA) targeting the oncogene was transfected into K562 cells.</p><p><b>METHODS</b>pAVGS4 (an eukaryocyte expression vector containing M1-GS RNA sequence) and pNAV-1 (as the control) were transfected into K562 cells by X-tremeGENE Q2. Total RNA was extracted at 24, 48, 72 and 96 hours after transfection. Then RT-PCR was done to compare the products at different time point. After collecting pAVGS4-transfected cells and the control cells at 48 and 96 hours after transfection, total protein was extracted and quantified. Change of P210 was determined by Western blot. Colony formation was analyzed at 96 hours after transfection.</p><p><b>RESULTS</b>RT-PCR based on transfected cells at different time point showed that the amount of bcr-abl mRNA began to decrease at 24 hours and reduced to 9.2% and 2.5% respectively at 48 and 72 hours after transfection. Western blot showed that the expression of P210 in the pAVGS4 group reduced to 10.4% of the control at 48 hours and 6.7% of the control at 96 hours after transfection. The inhibition rate of colony formation was 81.3% after K562 cells were transfected by pAVGS4.</p><p><b>CONCLUSION</b>pAVGS4 can efficiently destroy bcr-abl mRNA in K562 cells. The transcript level of bcr-abl mRNA was reduced with the time after transfection. The expression of P210 was decreased significantly at 48 and 96 hours after transfection. K562 cell colony formation was prominently inhibited.</p>
Subject(s)
Humans , Escherichia coli Proteins , Genetics , Fusion Proteins, bcr-abl , Genetics , Metabolism , Genetic Vectors , K562 Cells , RNA, Bacterial , Genetics , RNA, Catalytic , Genetics , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease P , Genetics , Time Factors , Transfection , MethodsABSTRACT
<p><b>OBJECTIVE</b>This paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression.</p><p><b>METHODS</b>It was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR.</p><p><b>RESULTS</b>It showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01).</p><p><b>CONCLUSION</b>M1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.</p>