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Optimization the expression of human papilloma virus E6 and E7 polytopic construct in E. coli expression system
Arian Rahimi , Arash Arashkia , Amir Mirzaie , Hassan Noorbazargan , Seyed Ataollah Sadat Shandiz , Roghayeh Rahimi , Mehdi Mahdavi ,
Volume 73, Issue 9 (December 2015)
Abstract

Background: Human papilloma virus is a DNA virus from the papillomavirus family that is most prevalent in human cervical cancers and many studies showed the E6 and E7 proteins are present in the majority of cervical cancer cases. Development of universal HPV peptide-based vaccine with more serotypes coverage has considerable value. The aim of the study was to design a multi-epitope universal vaccine for major HPV based on E6 and E7 proteins and optimization the expression of polytopic construct contains E6 and E7 genes from different genotypes of human papilloma virus as a candid vaccine.

Methods: In this experimental study that was carried out in Pasteur Institute of Iran, Virology Department from October 2013 to November 2014. In order to design the polytypic construct, we predicted the most probable immunogenic epitopes of E6 and E7 from common high risk HPV16, 18, 31, 45 along with high prevalent type 6 and 11 using bioinformatics methods. The synthetic pET28a expression vector harboring E6 and E7 protein was transformed into Escherichia coli hosts and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to expression optimization of recombinant protein, cell density, induction time, growth temperature, IPTG (Isopropyl &beta-D-1-thiogalactopyranoside) concentration and cultures media were studied.

Results: In the present study the recombinant fusion protein was expressed successfully and the highest expression of target protein was achieved in super broth medium containing 0.1% glucose and 0.2% L-arabinose. In Super broth medium, the optimum condition for recombinant protein expression was occurred at OD600 of 0.8, 0.1mM IPTG, one hour’s incubation time at 37 °C and BL21 (A1) host.

Conclusion: The results of this study show that the optimum expression of E6 and E7 proteins from different genotypes of human papilloma virus can be performed. Moreover, by purification of recombinant protein and evaluation of its immunogenicity in mice, it can be used as a vaccine candidate against the human papilloma virus.


Evaluation of chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effects of Aloysia citrodora extract on colon cancer cell line
Amir Mirzaie , Seyed Ataollah Sadat Shandiz, Hassan Noorbazargan , Elahe Ali Asgary,
Volume 74, Issue 3 (June 2016)
Abstract

Background: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer (HT29) cells by using real-time polymerase chain reaction and flow-cytometry methods.

Methods: This experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS) technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), disk diffusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, respectively. The half maximal inhibitory concentration (IC50) value was calculated. We extracted total RNA molecules by using RNX solution, after which cDNA was synthesized. Finally, the pro-apoptotic (Bax) and anti-apoptotic (Bcl2) gene expression was performed by real-time polymerase chain reaction and apoptotic effects were analyzed using Flow-cytometry method.

Results: GC-MS analysis of Aloysia citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57%) and Caryophyllene oxide (15.15%) The antioxidant activity of the extract was IC50= 0.6±0.03 mg/ml. The maximum and minimum antibacterial effects of extract were belonged to gram-negative and gram-positive bacteria, respectively. Cytotoxic results revealed that the A.citrodora extract have IC50= 20.1±0.78 mg/ml against colon cancer (HT29) cell line and real-time polymerase chain reaction results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470±0.72 (P< 0.05), 0.43±0.35 (P< 0.05)). In addition, the flow-cytometry results indicated the 38.66% apoptosis in colon cancer cell line.

Conclusion: According to the results, it seems that A. citrodora extract has potential antioxidant, antibacterial and anticancer effects and it suggested that further studies were performed for A. citrodora pharmaceutical importance.


Phytochemical composition, antibacterial and anticancer activities of Trifolium cherleri extract on lung cancer cell line (A549) and analysis of caspase 3 and caspase 9 apoptosis genes expression
Amir Mirzaie , Aliasghar Bagheri Kashtali , Hassan Sahebjamee , Hassan Noorbazargan , Hassan Rahmati , Seyed Ataollah Sadat Shandiz,
Volume 75, Issue 5 (August 2017)
Abstract
Background: Medicinal plants have been identified and used from prehistoric times and these plants make many chemical compounds for biological functions. Trifolium cherleri is an herbaceous species belonging to the family of the Fabaceae to Africa, Eurasia and Australia. T. cherleri is an important member of the Fabaceae family that is well-known herbal medicine in Iran. The aim of this study was to investigate the phytochemical composition, antibacterial and anti-cancer activities of T. cherleri extract.
Methods: This experimental study was performed in Islamic Azad University, from December 2016 to February 2017. At first, the phytochemical constituents of T. cherleri extract were determined using gas chromatography-mass spectrometry (GC-MS) method. Subsequently, the antibacterial activity of the extract was evaluated against some gram positive and negative pathogenic bacteria included Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615, Salmonella enteritidis ATCC 13076 and Listeria monocytogenes ATCC 35152 via minimum inhibitory concentration (MIC) method. Moreover, anticancer potential of extract was examined by colorimetric MTT assay toward lung cancer (A549) cell line. Then, the evaluation of caspase 3 and 9 apoptosis gene expression was determined using Real-Time Polymerase Chain Reaction (Real-Time PCR) technique. Moreover, the Real-Time PCR was performed using relative quantitative method.
Results: The phytochemical analyses of T. cherleri extract showed the 20 major components and the most frequent component was belonged to hexadecanoic acid, ethyl ester (20.7%) and 2-Pentadecanone, 6,10,14-trimethyl (19.9%). The extract had maximum antibacterial effects against Staphylococcus aureus and Streptococcus pyogenes. There was a dose dependent increase in the cytotoxicity effect of extract against A549 cancer cell. Moreover, the Real-Time PCR results indicated that the caspase 3 and caspase 9 gene expression was significantly up-regulated 2.57±0.27 (P<0.05), and 3.3±0.46 (P<0.05), respectively.
Conclusion: The results of this study showed that the T. cherleri extract had significant anti-bacterial and anti-cancer effects and it appear that the extract has potential uses for pharmaceutical industries. Moreover, it could be considered as a promising source for novel drug compounds, but more studies are needed.

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