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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Basic Medical Sciences
- نوع مقاله: Journal Article
- کلمات کلیدی: Tumor targeting,Mda-7,RGD peptide
- چکیده انگلیسی: Objective(s): Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression. Materials and Methods: mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA–cloning system. The colonies including these genes were selected by blue–white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids. Results: Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them. Conclusion: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.
- انتشار مقاله: 03-06-1394
- نویسندگان: Mahboobeh Khodadad,Seyed Younes Hosseini,Fatemeh Shenavar,Nasrollah Erfani,Samaneh Bina,Shahin Ahmadian,Mohammad-Reza Fattahi,Reza Hajhosseini
- جایگاه : پژوهشی
- مجله: Asian Pacific Journal of Cancer Prevention
- نوع مقاله: Journal Article
- کلمات کلیدی: Hepatitis C Virus,Drug resistance,Phylogenetic analysis,protease inhibitors,NS3
- چکیده انگلیسی: Background: Even with the fantastic successes of direct-acting antivirals (DAA) in the treatment of Hepatitis C
Virus (HCV) infection, natural drug resistance remains a challenging obstacle for their impacts. The data regarding
protease inhibitors (PIs) resistance in Iran population are limited. The aim of this study was to investigate the variations
in NS3 protease of HCV from non-responder patients. Methods: In this cross-sectional study, 14 HCV infected patients
with genotype 1(N=5) and 3(N=9) who have not responded to Interferon-related regime were enrolled from Liver
Clinic, Shiraz. The NS3 protease region was amplified by Nested-PCR followed by product gel extraction. Besides,
some amplified protease regions were cloned into a cloning vector to improve the sensitivity of mutation detection.
Both crude and cloned sequences were then introduced into sequencing. The obtained sequences were compared with
the NS3 reference sequences and analyzed by Geno2pheno available software to find possible substitutions. In the
end, the phylogenetic tree was constructed. Results: Among variations responsible for PIs resistance, only one out of
14 (7%) sample who was infected with genotype 1a, harbored R117C+N174S double mutation, which causes reduced
susceptibility to Telaprevir. Any another resistance mutation was not found among the studied population. The most
frequent substitutions were determined as I52M(N=9), S102A(N=9), S166A(8) and V170I(8) for genotype 3a, and
F147S/A(4) for genotype 1. However, some uncharacterized substitutions on scored position, including I132L(N=1),
I170V(N=3) and N174S(N=2) were also determined among sequences. Phylogenetic analysis demonstrated that the
protease region has enough power to correctly classify enrolled samples into relevant clusters on the tree. There were 2,
3 and 9 cases of sub-genotypes 1a, 1b, and 3a, respectively. Conclusion: A low frequency of PIs resistance mutations
in our HCV infected population is a hopeful point of starting these drugs in HCV infected patients.
- انتشار مقاله: 03-10-1397
- نویسندگان: Nargess Nejabat,Seyed Younes Hosseini,Jamal Sarvari,Ali Akbar Gorzin,Mohamad Reza Fattahi,Mohammad Rasoolian