Phage based vaccine: A novel strategy in prevention and treatment.

The vaccine was first developed in 1796 by a British physician, Edward Jenner, against the smallpox virus. This invention revolutionized medical science and saved lives around the world. The production of effective vaccines requires dominant immune epitopes to elicit a robust immune response. Thus, applying bacteriophages has attracted the attention of many researchers because of their advantages in vaccine design and development. Bacteriophages are not infectious to humans and are unlikely to bind to cellular receptors and activate signaling pathways. Phages could activate both cellular and humoral immunity, which is another goal of an effective vaccine design. Also, phages act as an effective adjuvant, along with the antigens, and induce a robust immune response. Phage-based vaccines can also be administered orally because of their stability in the gastrointestinal tract, in contrast to common vaccination routes, which are intradermal, subcutaneous, or intramuscular. This review presents the current improvements in phage-based vaccines and their applications as preventive or therapeutic vaccines.

The potential therapeutic impacts of trehalose on cardiovascular diseases as the environmental-influenced disorders: An overview of contemporary findings.

Cardiovascular diseases (CVDs) as environmental-influenced disorders, are a major concern and the leading cause of death worldwide. A range of therapeutic approaches has been proposed, including conventional and novel methods. Natural compounds offer a promising alternative for CVD treatment due to their ability to regulate molecular pathways with minimal adverse effects. Trehalose is natural compound and disaccharide with unique biological functions and cardio-protective properties. The cardio-protective effects of trehalose are generated through its ability to induce autophagy, which is mediated by the transcription factors TFEB and FOXO1. The stimulation of TFEB plays a significant role in regulating autophagy genes and autophagosome formation. Activation of FOXO1 through dephosphorylation of Foxo1 and blocking of p38 mitogen-activated protein kinase (p38 MAPK) also triggers autophagy dramatically. Trehalose has been shown to reduce CVD risk factors, including atherosclerosis, cardiac remodeling after a heart attack, cardiac dysfunction, high blood pressure, and stroke. It also reduces structural abnormalities of mitochondria, cytokine production, vascular inflammation, cardiomyocyte apoptosis, and pyroptosis. This review provides a molecular overview of trehalose's cardioprotective functions, including its mechanisms of autophagy and its potential to improve CVD symptoms based on clinical evidence.

Crosstalk of Transcriptional Regulators of Adaptive Immune System and microRNAs: An Insight into Differentiation and Development.

MicroRNAs (miRNAs), as small regulatory RNA molecules, are involved in gene expression at the post-transcriptional level. Hence, miRNAs contribute to gene regulation of various steps of different cell subsets' differentiation, maturation, and activation. The adaptive immune system arm, which exhibits the most specific immune responses, is also modulated by miRNAs. The generation and maturation of various T-cell subsets concomitant with B-cells is under precise regulation of miRNAs which function directly on the hallmark genes of each cell subset or indirectly through regulation of signaling pathway mediators and/or transcription factors involved in this maturation journey. In this review, we first discussed the origination process of common lymphocyte progenitors from hematopoietic stem cells, which further differentiate into various T-cell subsets under strict regulation of miRNAs and transcription factors. Subsequently, the differentiation of B-cells from common lymphocyte progenitors in bone marrow and periphery were discussed in association with a network of miRNAs and transcription factors.

Gene Editing-Based Technologies for Beta-hemoglobinopathies Treatment.

Beta (ß)-thalassemia is a group of human inherited abnormalities caused by various molecular defects, which involves a decrease or cessation in the balanced synthesis of the ß-globin chains in hemoglobin structure. Traditional treatment for ß-thalassemia major is allogeneic bone marrow transplantation (BMT) from a completely matched donor. The limited number of human leukocyte antigen (HLA)-matched donors, long-term use of immunosuppressive regimen and higher risk of immunological complications have limited the application of this therapeutic approach. Furthermore, despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as nanomedicine and gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, including transcription activator-like effector nucleases, zinc-finger nucleases, and clustered regularly interspaced short palindromic repeat-Cas-associated nucleases. These tools have concentrated on γ- or ß-globin addition, regulating the transcription factors involved in expression of endogenous γ-globin such as KLF1, silencing of γ-globin inhibitors including BCL11A, SOX6, and LRF/ZBTB7A, and gene repair strategies. In this review article, we present a systematic overview of the appliances of gene editing tools for ß-thalassemia treatment and paving the way for patients' therapy.

Conceptual Framework for SARS-CoV-2-Related Lymphopenia.

The emerging of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak is associated with high morbidity and mortality rates globally. One of the most prominent characteristics of coronavirus disease-19 (COVID-19) is lymphopenia, which is in contrast to other viral infections. This controversy might be explained by the evaluation of impaired innate and adaptive immune responses, during the SARS-CoV-2 infection. During the innate immune response, poly-ADP-ribose polymerase hyperactivated due to virus entry and extensive DNA damage sequentially, leading to nicotinamide adenine dinucleotide (NAD)+ depletion, adenosine triphosphate depletion, and finally cell death. In contrast to the immune response against viral infections, cytotoxic T lymphocytes decline sharply in SARS-CoV-2 infection which might be due to infiltration and trapping in the lower respiratory tract. In addition, there are more factors proposed to involve in lymphopenia in COVID-19 infection such as the role of CD38, which functions as NADase and intensifies NAD depletion, which in turn affects NAD+-dependent Sirtuin proteins, as the regulators of cell death and viability. Lung tissue sequestration following cytokine storm supposed to be another reason for lymphopenia in COVID-19 patients. Protein 7a, as one of the virus-encoded proteins, induces apoptosis in various organ-derived cell lines. These mechanisms proposed to induce lymphopenia, although there are still more studies needed to clarify the underlying mechanisms for lymphopenia in COVID-19 patients.

The Molecular Basis of COVID-19 Pathogenesis, Conventional and Nanomedicine Therapy.

In late 2019, a new member of the Coronaviridae family, officially designated as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

MicroRNAs as the actors in the atherosclerosis scenario.

Atherosclerosis is considered as the most common cardiovascular disease and a leading cause of global mortality, which develops through consecutive steps. Various cellular and molecular biomarkers such as microRNAs are identified to be involved in atherosclerosis progression. MicroRNAs are a group of endogenous, short, non-coding RNAs, which are able to bind to specific sequences on target messenger RNAs and thereby modulate gene expression post-transcriptionally. MicroRNAs are key players in wide range of biological processes; thus, their expression level is regulated in pathophysiological conditions. Ample evidences including in vitro and in vivo studies approved a critical role of microRNAs in epigenetic and the sequential processes of atherosclerosis from risk factors to plaque formation, progression, and rupture. Based on these findings, miRNAs seems to be promising candidates for therapeutic approach. This review summarizes the role of miRNAs in atherosclerosis development, epigenetic, and therapy. Moreover, the application of exosomes in miRNA delivery, and/or their prognostic and diagnostic values are also discussed.

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis.

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K d ) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.

Engineered zinc-finger nuclease to generate site-directed modification in the KLF1 gene for fetal hemoglobin induction.

Elevation of hemoglobin F (HbF) ameliorates symptoms of ß-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to ß hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in ß-thalassemia patients.

Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F' was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.

Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin.

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.

Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of ß-thalassemia.

Elevation of Hemoglobin F ameliorates symptoms of ß-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to ß-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for ß-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

MicroRNA: Relevance to stroke diagnosis, prognosis, and therapy.

Stroke is a life-threatening disease that accounts for a considerable burden of mortality in both developing and developed world. Identification of specific biomarkers for stroke and its outcomes can greatly contribute to improved care of patients. MicroRNAs (miRNAs) are known as novel biomarkers that could be used as diagnostic, prognostic, and therapeutic biomarkers. Various studies have shown that miRNAs have key roles in the pathogenesis of stroke, and its complications and outcomes. In addition, there is evidence showing that mesenchaymal stromal cell-derived exosomes containing miRNAs can be used for monitoring and treatment of various diseases such as stroke. Here, we summarized various aspects of miRNA applications in different stages of stroke.

Plasminogen Activator Inhibitor Type-1 as a Regulator of Fibrosis.

Fibrosis is known as a frequent and irreversible pathological condition which is associated with organ failure. Tissue fibrosis is a central process in a variety of chronic progressive diseases such as diabetes, hypertension, and persistent inflammation. This state could contribute to chronic injury and the initiation of tissue repair. Fibrotic disorders represent abnormal wound healing with defective matrix turnover and clearance that lead to excessive accumulation of extracellular matrix components. A variety of identified growth factors, cytokines, and persistently activated myofibroblasts have critical roles in the pathogenesis of fibrosis. Irrespective of etiology, the transforming growth factor-ß pathway is the major driver of fibrotic response. Plasminogen activator inhibitor-1 (PAI-1) is a crucial downstream target of this pathway. Transforming growth factor-ß positively regulates PAI-1 gene expression via two main pathways including Smad-mediated canonical and non-canonical pathways. Overexpression of PAI-1 reduces extracellular matrix degradation via perturbing the plasminogen activation system. Indeed, elevated PAI-1 levels inhibit proteolytic activity of tissue plasminogen activator and urokinase plasminogen activator which could contribute to a variety of inflammatory elements in the injury site and to excessive matrix deposition. This review summarizes the current knowledge of critical pathways that regulate PAI-1 gene expression and suggests effective approaches for the treatment of fibrotic disease. J. Cell. Biochem. 119: 17-27, 2018. © 2017 Wiley Periodicals, Inc.

The silencing effect of miR-30a on ITGA4 gene expression in vitro: an approach for gene therapy.

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4ß1 and lymphocyte Peyer's patch adhesion molecule-1 or α4ß7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 (ITGA4) is overexpressed in some immune disorders and cancers. Thus, inhibition of ITGA4 could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress ITGA4 expression. The ITGA4 3' UTR was amplified, cloned in the Z2827-M67-(ITGA4) plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the ITGA4 expression analysis. The findings of this study showed that miR-30a downregulated ITGA4 expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the ITGA4 gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy.

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

BACKGROUND: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. MATERIALS AND METHODS: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. RESULTS: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001). CONCLUSION: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Evaluation of Urokinase Plasminogen Activator Receptor, Soluble Urokinase Plasminogen Activator Receptor, and ß1 Integrin in Patients with Hodgkin's Lymphoma.

BACKGROUND: The objective of this study is to indicate the role of urokinase plasminogen activator receptor (uPAR), soluble uPAR (suPAR), and ß1 integrin in tumor growth and invasion of lymph nodes from Hodgkin's lymphoma (HL) patients. MATERIALS AND METHODS: In this study, 25 lymph nodes from HL patients were analyzed for the expression of ß1 integrin and uPAR on mononuclear cells using two-color flow cytometry and immunohistochemical analysis. Moreover, the levels of suPAR in the serum samples of HL patients were measured and compared with 32 healthy controls. RESULTS: Flowcytometry and immunohistochemical results indicated no significant association of uPAR expression with tumor size, different stages, or different histological subtypes of HL; however, an increased expression of ß1 integrin was detected in the advanced stages of HL. Higher expression of ß1 integrin was detected in nodular sclerosis compared to lymphocyte predominant. No significant difference was observed between the serum levels of suPAR in patients with different stages of HL and healthy controls. Moreover, the levels of suPAR were significantly higher in nodular sclerosis in comparison with other subtypes. CONCLUSIONS: This study showed that the levels of suPAR and ß1 integrin varied between different histological subtypes of HL. Although uPAR may play only a minor role in the growth and metastasis of lymphoma, ß1 integrin may be important in predicting prognosis and metastasis in HL.

The Increase in Protein and Plasmid Yields of E. coli with Optimized Concentration of Ampicillin as Selection Marker.

Background:Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 µg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 µg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109 , 3.21×109 , 2.32×1010 , 8.11×108 , respectively. The plasmid yields were 55 ng.µL-1, 69 ng.µL-1, 164 ng.µL-1 and 41 ng.µL-1, respectively. Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 µg.mL-1) was significantly (p < 0.01) higher than other doses.