فهرست مطالب مهدی زین الدینی

Gold nanoparticles (GNPs) with unique optical properties, such as easy operation and visualized assay, have a great ability to detect different types of analytes. Today, the use of gold nanoparticles has wide applications in the field of medicine and biotechnology, including the detection of microorganisms that cause contamination in water, air and food and it is considered a suitable alternative for chemical and physical methods. New technologies in the design of biosensors based on GNPs provide the ability to identify biological compounds accurately and quickly. One of these technologies is a detection sensor based on surface plasmon resonance (SPR), which based on its optical properties, is capable of very sensitive and specific measurement of biomolecule interactions without time delay. This technology can quantify in a short time the properties of biomolecular mediators (such as oligonucleotides, proteins and bacteria) on the surface, including reaction speed, tendency and concentration of surface mediators. In this review, while investigating the surface plasmon properties of gold nanoparticles, the simple diagnostic applications of gold nanoparticles based on the localized surface plasmon (LSPR) method and detection in biomedicine.

Armed attacks on military and civilian targets to achieve political expediency have long been a means of using force and power. This matter has attracted international attention for almost a century in the Palestinian land with the violence of the occupying Zionist regime of Israel against the Palestinian civilians. However, no international reaction from the Arab Muslim countries and the actions of the United Nations have been able to prevent the invasion and occupation of the oppressive Israeli regime. This has become an important factor in mobilizing the global public opinion of the nations against the heinous crimes against humanity of this regime. Israel's attacks on the Gaza Strip after October 7, 2023, for nearly four months continuously, have left more than tens of thousands of civilian dead in Gaza, while civilians are immune from military attacks by the warring parties according to the international rules of humanitarian law. . The parties, whether in the position of attack or in the position of defense, must support civilians according to the fundamental principles of the aforementioned rules. Despite these principles, committing massive human atrocities in the Gaza Strip against women and children has shown the failure of international law rules. And the criminal regime is murdering women and children by suggesting that the civilians of Gaza are no longer civilians with the help and assistance of the Hamas militant group and can be the target of military attacks. Now, in this article, by raising the question: Can the Zionist regime of Israel make Palestinian civilians a military target under the pretext of supporting the Hamas militant group? The necessary investigations are carried out with a descriptive and analytical method in relation to the fundamental principles of humanitarian rights. to show the violations of these rules by the Zionist regime of Israel.

Aptamers or chemical antibodies are mostly nucleic acid sequences that can bind to various diverse targets including small molecules, large molecules, and cells. Among the advantages of aptamers compared to antibodies, we can mention the in vitro production process, the possibility of choosing aptamers against toxic and non-immunogenic molecules, long-term storage, and much lower production costs. Aptamers can also be easily modified or stabilized, and their synthesis is associated with high purity and reproducibility, they are chemically stable, and due to their nucleic acid structure, they are much more flexible than antibodies, and they can be used in the form of molecular beacons probes, a combination of use aptamer-target interaction and nucleic acid amplification to achieve highly sensitive detection ranges. These interesting features have made aptamers ideal diagnostic elements for analytical tools such as biosensors, colorimetric methods, surface plasmon resonance, and lab-on-a-chip. However, all these methods require skilled workers and laboratory-based instruments, thus limiting their application. In this regard, lateral flow assay or paper strip kits provide fast and reliable results and only require user intervention in the sample addition phase. Due to their simplicity is widely used in various fields including medicine, food product quality inspection, and environmental safety. In this work, while introducing aptamers, , an overview of its unique application in lateral flow assay and the future of this technology should be studies.

Gene editing as a molecular knife has provided a powerful tool to edit the genome inside living organisms’ bodies. Despite all the benefits, this technology has many biosecurity concerns that have prompted the international community to develop specific implementation guidelines. This study aimed to investigate the risks of gene editing technology from the point of view of biosecurity and to determine its defense strategies.

This review was conducted in the fall and winter of 2022-23. The study’s method was based on the observation and interpretation of the data obtained from relevant scientific articles and books. Scientific books and articles were searched by searching the keywords Germline Gene Editing, CRISPR, Biosecurity, CRISPR War, Biohacking, CRISPR babies, Do It Yourself, Islamic Bioethics in PubMed, Scopus, Researchgate databases and also the Google search engine was searched and checked in English .

The advent of genome editing technology has created a new paradigm in which the human genome sequence can be precisely manipulated to achieve a therapeutic effect. However, embryo editing and the design of programmed humans (super-humans) are considered one of the challenges and risks of gene editing biosecurity. Also, this technology is mentioned as a dangerous tool for biohacking and bioterrorism in the design of personalized bioweapons and emerging agents.

CRISPR-based bioweapons have destroyed the logical and strategic balance of power that has kept the world immune from using weapons of mass destruction. The world is facing a potentially more dangerous technology than nuclear weapons. As a result, establishing appropriate international and ethical laws is necessary to prevent the potential dangers of this technology and to deal with it.

Aflatoxin B1 is a type of mycotoxin produced by Aspergillus fungi during food production and storage. Aflatoxins have many toxic effects on the body that cause mutagens, teratogens and have high carcinogenic properties that cause cancer in the liver and other organs. Although conventional device methods for measuring aflatoxin B1 in food are sensitive and accurate, they have disadvantages such as high diagnostic time, high cost, the need for a trained user, and the creation of false positive results. Therefore, the development of new measuring methods has been prioritized by researchers. Among these measurement methods is the use of biosensors, which are fast, simple and more affordable and are used in the food industry today. In this work, a colorimetric optical aptasensor using gold nanoparticles with appropriate sensitivity and high selectivity was used to detect aflatoxin B1 in serum and buffer. For this purpose, gold nanoparticles were synthesized by reducing HAuCl4 by sodium citrate (with a size of 14.40 nm and a zeta potential of -27.5). In this method, the protective effect of DNA sequence on the surface of gold nanoparticles has been used in the presence or absence of aflatoxin with the intervention of salt and the characteristic of visual color change. The detection limit of this method was estimated to be 50 ng/L and its linear range was 200-28000 ng/L. As a result, the designed aptasensor can be used for quick identification and screening of this toxin in contaminated food.

Shigellosis is an infectious disease caused by Shigella dysenteriae and threatens the lives of millions of people worldwide annually. IpaB protein, which is part of the Shigella type III secretion system (T3SS), it can elicit a favorable immune response against the bacterium. The aim of this study is experimental evaluation of the immunogenicity of a recombinant protein containing immunogenic regions of IpaB as a subunit recombinant vaccine candidate against Shigella dysenteriae.

The gene encoding immunogenic protein that cloned into pET28a expression vector was transformed in the bacterial host E.coil strain Rosetta (DE3) and induced by IPTG. The purified protein was achieved using nickel chromatography column and injected into guinea pigs for immunization. The produced antibody titer was assessed by indirect ELISA assay and finally animal challenge was performed using the Sereny test in guinea pigs.

A 36 KDa band as IpaB protein was observed in SDS-PAGE which was confirmed by western blot. The immunological analyses showed production of high titer of specific anti-IpaB antibody (1:102,400) in immunized Guinea pig. The absence of any keratoconjunctival inflammation in immunized guinea pigs in Sereny test indicated high level protection against  virulent Shigella dysentriea.

The results showed that IpaB can elicit high titer of antibody and protection against Shigella dysentriae in Guinea pigs.

The Coronavirus Disease 2019, named COVID-19 is a global problem. According to the declaration of the World Health Organization (WHO), it is a novel and extreme outbreak, spreading worldwide. Firstly, numerous patients reported exposure in Wuhan City, China at a large animals and seafood market. Accordingly, the first idea is suggesting the animal-to-human transmission of this infection pathogen. Next, since then, numerous patients have decelerated no exposure to animal shops, indicating that human-to-human transmission is occurring. The new coronavirus (2019-nCoV) is a positive RNA virus with a 29.8 kb genome and S, E, M, N, and Orf1 gene fragments. The most popular method for 2019-nCoV detection is genome-based approaches, like Polymerase Chain Reaction (PCR)-based tests that require expensive experimental equipment, a controlled working environment, and high-trained technicians; they are often lacking in massive viral outbreaks. Therefore, another rapid and simple genome-based assay was evaluated for this pathogen. In this review study, the RT-LAMP technique, as the main isothermal amplification assays with less time consumption and without the need for expensive equipment, compared to conventional PCR-based methods to 2019-nCoV identification, was discussed. Accordingly, the advantage or disadvantage of these techniques was compared. The obtained data indicated that this molecular and isothermal method could be used as a successful one-step process for portable screening and the rapid identification of 2019-nCoV.

In microbial contamination of food and water, identification of trace amounts of contaminating bacteria has always been an interest and concern for researchers. The most common way to remove of this problem is culture-based methods in order to increase and enrichment of bacteria samples, which extends the bacterial detection process to several hours or days. One of the smart strategies to solve this problem is concentrated of bacteria using physical methods. The aim of the present study was to enrich of Vibrio cholerae as the most important water polluting germs using filtration method and evaluation of its function by culture method and two detection methods ATP and PCR assay.

A certain concentration of V. Cholerae was added into a specified volume of sterile water, artificially. Then, the bacteria were extracted from the medium and filtered using 0.450µm separable filters. Finally, the performance of the pre- and post-filtration process was compared using bacterial cell culture (CFU), ATP and PCR assay with the specific primers for the ompW gene of V. cholerae.

The results of this work showed that this method shows high efficiency and recovery performance. So that, samples had no positive response before filtration in both methods, but after filtration in isolated and recovered samples, the presence of bacteria was detected in both ATP and PCR methods.

In conclusion, this strategy can detect V. cholerae in non-culture and in the least amount of time in contaminated water samples.

Aim and

In the past two decades, proteins and peptides have become an important class of therapeutic drugs. However, their sensitivity to chemical and physical degradation has posed challenges for scientists. Approximate 90% of the chicken feather of slaughterhouse waste is Keratin. Keratin protein is insoluble in water and has a strong tendency to accumulate and precipitate. Today, the stability of proteins in pharmaceutical applications is of great importance.

In this study, Keratin was extracted from feather by sodium hydroxide 0.2 M and hydrolyzed by 8% (V/V) Alcalase enzyme in 55 Celsius, at 8 pH. The hydrolyzed Keratin solution was chemically modified by alkylation and sulfitolysis to reduce aggregation. Keratin solubility, degree of chemical modification, percentage of stability, mol amount of free thiol, also DLS, and FTIR of the samples were investigated.

The result showed that the degree of chemical modification of Keratin in the alkylation method was 72% and the sulfitolysis method was 66%. The chemical modification of hydrolyzed Keratin was reduced aggregation at pH3 to 288.65% and 335.3% for alkylation method and sulfitolysis respectively. Investigation of the stability and preservation of protein content showed that the stability of modified Keratin by alkylation method was 81.9% and by sulfitolysis method was 86.3% in 30 days, but the hydrolyzed Keratin as a control preserved only 9.8% of protein content. The result of the DLS test shows that the Keratin modified by alkylation in water has PI=0.476 and a mean particle size of 157.3 nm and Keratin modified by sulfitolysis in water has PI=0.475 and a mean particle size of 136.2 nm, which was more uniform and dispersed than the hydrolyzed Keratin with PI=0.552 and a mean particle size of 990.3 nm.

The chemical modification of hydrolyzed Keratin was a permanent change and binding of expected groups increased the Keratin solubility in aqueous solution and decreases the aggregation at acidic pH. The results of the FTIR analysis confirmed the chemical modification of Keratin.

Following the emergence of COVID-19 caused by the SARS-CoV2, the reasons for the emergence of the novel virus have been the subject of interest for molecular biology researchers and news agencies. This article attempted to emphasize all aspects of the emergence of this virus and discuss the latest information related to its development.

From the main site for articles published on COVID-19 (LitCovid), more than 34000 articles have been published, until July 22. But there have been been no scientific discussion about the reasons for the emergence of the virus due to its natural origin or genetic manipulation in research laboratories. In this review study, by collecting and reviewing 15 articles related to this topic, the scientific reasons for the emergence of this virus is analyzed.

Reverse genetic technology is a powerful tool for genetic instructions and design of engineered viruses to produce effective drugs and vaccines against viral infections. But dangerous viruses may also be created in this way that exhibit unknown properties. Studies have shown that SARS-CoV2 has a unique sequence in the middle of the gene encoding glycoprotein Spike (S) that is not similar to other coronaviruses.

Due to the specific features of SARS-CoV 2 and its comparison with other coronaviruses, its emergence and novel agents has been determined in the scientific community. For this, laboratory production and genetic manipulation are possible, but whether its occurrence is accidental or intentional requires further investigation.

Today, one of the most important problems in detection of human pathogens, is lack of positive control. The idea of using hybrid vectors, containing genes of different pathogens, can overcome this limitation. We can design specific primers for each region and use the hybrid vector as positive control sample in PCR. In this research we designed an in silico hybrid vector and relevant primers for detection of Francisella, Variola, Burkholderia and Yersinia.

In this study, fopA, caf1, 16srRNA and HA genes were chosen to be located on the vector, to respectively represent Francisella, Yersinia, Burkholderia and Variolla,. The sequence of these genes were obtained from NCBI in FASTA format and were aligned in BioEdit 7.0.5.3 software for finding conserve region of each gene, then some purposeful changes were applied in the sequence of each gene and the sequences were placed next to each other and the construct was designed. Specific primers were designed for each region using Oligo7, BioEdit, OligoAnalyzer tool as well as NCBI database. Finally, the construct was cloned in PUC57 in SnapGene 3.2.0.1 and PCR was simulated on hybrid vector using designed primers.

Analysis confirmed that conserved regions for each gene were located on hybrid vector, and simulation of PCR proved the accuracy of designed primers.

A genetic simulator hybrid construct with the types of primers required to identify the four factors Francisla, Varivola, Borghuleria and Yersinia has been designed in silico so that such factors could be identified under positive control in emergencies.

This study aimed to produce recombinant hybrid protein, DT389GCSF, in E. coli BL-21(DE3) in a soluble and active form and to purify it and evaluate its cytotoxicity.

The protein, His6-tagged DT389GCSF was expressed in E. coli BL-21(DE3) in a soluble form at 28 °C. Then it was purified by affinity chromatography on nickel sepharose column. Finally, the function of purified recombinant protein was evaluated by MTT assay on HL-60 cancer cell line.

The yield of expression and purification of DT389GCSF were determined at about 12 and 80 percent, respectively, using Image J software. The IC50 value upon 48 hours of exposure of DT389GCSF toward cell line was about 0.00017±0.000019 M.

By reducing the temperature and using the intracellular mechanism, the refolding of hybrid protein, DT389GCSF, can be observed in a proper and active form. As a result, in vitro production of relevant immunotoxins, by using this method, the steps of inclusion body production can be neglected.

Ricin toxin is a heterodimeric glycoprotein consists of two polypeptide chains, A and B, joined by a disulfide bond. Ricin poisoning can occur via ingestion, inhalation, or injection. Immunogenicity studies are now focused on two RTA-based subunit vaccine candidates, including RiVax and RVEc. These studies have examined the vaccine candidate immunization as an alone and in combination with adjuvant, however, there is not a published study on the immunogenicity evaluation of the candidate vaccine through the delivery by nanoparticles. The aim of this study was preparation and evaluation of RiVax Recombinant Vaccine-loading in chitosan nanoparticles.
In this experimental study, after purification of the protein by Ni-NTA chromatography column, the protein was loaded with Ionic gelation method in chitosan nanoparticles. Then physicochemical properties of nanoparticles containing RiVax and stability of this protein were evaluated during the process of loading in the chitosan nanoparticles.
Findings: The results of this study showed that the nanoparticles containing protein had a size of 178 nm and a Zeta potential of .8 MV and a PDI of 0.193. It was also found that the RiVax recombinant protein was denatured during the process of preparing the chitosan nanoparticles.
The results of this study showed that the RiVax protein has been unstable in acidic conditions for the production of chitosan nanoparticles and this method is not suitable for loading this protein in chitosan nanoparticles.

Water pollution is one of the most important problems for human beings. BTEX (Benzene, Toluene, Ethylbenzene and Xylenes) have vast application in industry and their carcinogenic effect on human body has approved. Hence these part of water pollutants (water-soluble aromatic components) have more importance. Monitoring systems that can detect presence of BTEX in water supplies are much expensive such as gas chromatography so we need simple systems to reduce the number of samples that we are suspected to them for more analysis by much more expensive systems. Bioreporters are a subgroup of biosensors which are using for sensing and monitoring some signals or reagents. A bioreporter is an organism like a bacteria or a plant that is genetically manipulated to have a promoter which is sensitive to a chemical or physical signal. Activation of the promoter In presense of the signal leads to product of the reporter gene which can be sensed or calculated by our laboratory supplies. A green fluorescent protein gene has been used as a reporter gene downstream of PtbuA1 as a BTEX sensitive promoter in Escherichia coli and its response to BTEX has been investigated in this study. Our results show our bioreporter can sense Toluene.The optimum time and temperature for the bioreporter is also defined.

Elastin like protein or ELP is a synthetic biopolymer consisting the pentapeptide repeats of VPGXG (X can be any amino acid except Pro). This protein is the thermal responsive polypeptide that undergoes a reversible phase transition. At a temperature below the transition temperature (Tt), ELP molecules assume an extended conformation and thus are soluble in aqueous solution; but upon the temperature shift higher than the Tt, however, ELPs become insoluble and form the segregated phase prone to ‘aggrigate’ form. The aim of this study was cloning, expression and activity of ELP60 according to Tt.
Firstly, ELP10 (150 bp) was synthesized and cloned into pBluescript. Then, Elp60 (900 bp) was produced using recursive directional ligation (RDL). Finally, ELP60 was subcloned into modified pET25 and ELP60 expression was confirmed using SDS-PAGE method. Also, ELP60 were purified according to Tt and performance of inverse transition cycle (ITC), which confirmed its activity.
The results were shown that the pET-ELP60 constructed and ELP60 expressed; it was successfully purified (about 90%) in one step and non-chromatographic method.
From produced recombinant protein can be used for simple and easy purification of the recombinant protein as pharmaceutical drug, smart drug delivery and tissue engineering.

Ricin toxin is a disulfide-linked heterodimeric glycoprotein consisting of the toxic A chain and the cell-binding B chain that acts through the irreversible inactivation of eukaryotic ribosomes and inhibition of the polypeptide chain elongation, which finally leads to cell death. The toxin is extracted from castor plant beans (Ricinus communis) and it consists of 1–5% of the beans’ dry weight. Ricin is of concern in the area of biodefense because it is readily available to individuals or groups with little technical expertise or funding and its source is ubiquitous in the weed and in the cultivated crop in many countries. Since ricin can easily be distribute in the body, it has been classified by the CDC (US center for Disease Control and Prevention) as a class B biothreat. There has been much investigation on the diagnosis, treatment and production of a potent vaccine against this agent. For diagnosis of ricin poisoning in clinical cases, immunological techniques have been used for screening of samples and combination of high tech chromatographic and mass spectrometric methods could be used for poisoning confirmative analysis. There is currently no specific treatment available for poisoning with ricin but two closely related RTA-based subunit vaccines, contain RiVax and RVEc, are now under development. The aim of this study is presentation of ricin toxin, diagnosis methods, treatment and new vaccine candidates against it.

Current study is aimed at designing and producing gold-nanoparticle based aptasensor for colorimetric detection of tri-nitro toluene (TNT).
Gold-nanoparticles have a wide range of application in designing biosensors for their special and highly sensitive plasmonic characteristics. In the present study, TNT molecule has been detected by designing and producing gold nanoparticle based aptasensor. After synthesizing gold nanoparticles and functionalizing them by ssDNA aptamer having 52 nucleotides, aptasensor performance for detecting target molecule was evaluated. Techniques such as dynamic light scattering (DLS), transmission electron microscope and UV-Vis spectrophotometer were used to characterize the size and shape of nanoparticles, functionalizing them by aptamer, and also their sensitivity to detect target.
UV-Vis spectrophotometer, DLS, and TEM microscopy tests indicated that gold nanoparticles were synthesized with excellent quality and average size of 40 nm. TNT molecule was colorimetric detected with sensitivity of 15 nanomolar using gold nanoparticle based aptasensor.
According to the results it can argue that the strategy promises a novel method in detection due to wide range of such aptasensors detection. .

Brucellosis is a common disease between humans and animals. Vaccination is the best approach to prevent brucellosis. Existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. Therefore, it is necessary to generate an appropriate vaccine that is produced by molecular methods. The aim of this study was designing and construction of pDS132-Δkanr suicide vector to delete kanamycin resistance sequence (kanR) in a mutant strain of Brucella melitensis rev1 to generate a candidate vaccine strain.
In this study, the plasmid pDS132 was used as a suicide vector to delete target gene. In order to construct the deletion cassette, two homologous fragments were separately designed and constructed by PCR, and tandemly cloned into the pBluescriptSK(-) vector using appropriate restriction enzymes. Then, the deletion cassette was digested from the recombinant vector by terminal restriction enzymes, and sub cloned into the pDS132 vector to construct the suicide vector pDS132-ΔkanR.
The pDS132-ΔkanR contains 590bp upstream sequences and 421bp downstream sequences on the deletion cassette fragment; it can be used as specific suicide vector to disrupt the kanR sequence in genome of mutant strain of Brucella melitensis Rev1.
The use of suicide pDS132-ΔkanR system facilitated mutant construction which is a more specific and effective system in comparison with the other positive marker-dependent suicide systems and primary techniques such as serial passages.

Immunotoxin (IT) is a directed toxin containing two distinct sections (immune and toxin parts) covalently bond using specific chemical or peptide linkers. The aim of this study is to produce a recombinant and hybrid protein containing diphtheria toxin (DT) and granulocyte colony stimulating factor (G-CSF).
According to the structure of the first commercial recombinant immunotoxin (Ontak, hybrid protein containing DT fused interlukine2), gene encoding of DT and G-CSF was amplified using specific primers and plasmid template of pET-IDZ3 (pET21 harboring the gene encoding ontak immunotoxine) and pET-GCSF (pET23 harboring G-CSF), respectively. The DT-GCSF fusion protein produced using soeing PCR and specific primers. Finally, pET-DT-GCSF construction prepared using subcloning of DT-GCSF into pET21a() and confirmed by sequencing, SDS-PAGE and western blot technique.
Gene encoding of DT-GCSF inserted into NdeI/EcoRI site of pET21 and the construction of strain producing DT-GCSF recombinant immunotoxin was confirmed using customary methods.
The cytokine fusion protein, DT-GCSF, could be used for the inhibition of G-CSF receptor bearing cancer cells.

Inteins (INT) are internal parts of a number of proteins in yeast and some other unicellular eukaryotes, which can be separated from the immature protein during protein splicing process. After identifying the mechanism of intein action, applications of these sequences are be considered in the single- step purification of recombinant proteins and different intein tags were developed. The most important advantage of using intein tags in purification of recombinant proteins than other affinity tags is no requirement of expensive protease enzymes and following additional steps to remove protease that make intein tags economically are considered more important. In the present study, denileukin diftitox immunotoxin (brand name Ontak), be fused with an intein tag and it was inserted in pTXB1 plasmid.
In this study, with respect to multiple cloning sites (MCS) of pTXB1, specific primers were designed. Polymerase Chain Reaction (PCR) was performed and encoding sequence of ONTAK was cloned using restriction sites of NdeI and SapI. Recombinant vector (PTX-IDZ) was transformed into E. coli strain ER2566 and expression of gene was studied.
The accuracy of recombinant construct was confirmed by PCR and enzymatic digestion. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting.
Restriction site of SapI guarantees no additional residues incorporate in primary protein sequence. Also, the expression of this construct was analyzed in compare with fused protein to poly-His tag. According to the appropriate expression of fused protein in both constructs it was expected that one step- purification of considered drug protein will be success in the following steps.

The photoprotein aequorin, isolated from the jellyfish Aequoera victoria, is a calcium-sensitive bioluminescent protein formed with the apoprotein apoaequorin and the prosthetic group coelenterazine that emits light upon calcium binding. The protein is believed to be a monomer containing four helix-loop-helix motifs (EF hand) of which three domains can bind calcium. On the other hand, excess calcium in brain is associated with many neurodegenerative diseases and conditions including Alzheimer’s, Parkinson’s, and stroke. Aequorin as a natural calcium binding protein taking up excess calcium in brain cells much like a sponge. The aim of this study is production of mutant aequorin for calcium sensitivity increase.
Material and The mutated aequorin (N28D, K30T), in EF hand I, were prepared using site directed mutagenesis and subcloned in the pET21 expression vector. According to design of His-taq, purified mutants aequorin prepared using nickel affinity chromatography. Finally calcium sensitivity compared between wild-type and mutant aequorin.
Calcium sensitivity of mutated aequorin indicated aeq-K30T is more sensitive than wild type and aeq-N28D.

Immunotoxin is a fusion protein containing two distinct sections covalently bonded by specific linkers. It includes an immunological section with a diagnostic role to connect the immunotoxin to the specific receptors and a toxic section with a cytotoxic role that leads to the cell death. The aim of this study is development of a lymphoma cancer protein therapy method using a recombinant immunotoxin produced by connecting diphtheria toxin to human interleukin 2 (IL2). After the synthesis of the fusion protein gene sequence (IDZ: DT-IL2)، subcloning was done in the pET expression system. The pET-IDZ plasmid was transformed to BL21 (DE3) bacteria and then was induced. The protein purification was accomplished by nickel affinity chromatography system and then the function of the produced recombinant fusion protein was evaluated on K-562 cancerous cell line by MTT biological assay. After production of pET-IDZ plasmid، the expression of the recombinant fusion protein in BL21 (DE3) bacteria was validated using electrophoresis and Western Blot methods. The purification yield determined above 95% using densitometry method. Afterwards، K-562 cells were treated by different concentrations of the produced fusion protein. The results showed the proper function of the produced fusion protein. Also، IC50 % amount (immunotoxin concentration to remove 50% of the cells) was determined as 6×10-6 M. Immunotoxin or targeted toxin is a novel strategy for cancer patients. The first recombinant immunotoxin (named Ontak)، in which diphtheria toxin had been connected to interleukin 2، was approved from United States Food and Drug Administration (FDA) in 1999. Considering the importance of this drug، recombinant fusion protein was attempted to be produced in this research. Experimental data confirmed effective protein function، but complementary studies will be required to be compared with Ontak.

Nowadays photoproteins are excellent reporter systems as they have virtually no background. The most commonly studied photoprotein is aequorin. But because of it’s low quantum yield and very fast reaction kinetic; this photoprotein assay don’t adapt for High-Throughput Screening (HTS). Consequently, some researchers developed three improved photoproteins optimized for the generation of precise Ca2+ mobilization assays; photina, i-photina and c-photina. Although the total light release of these photoproteins is greater and their reaction kinetic is slower compared to other existing photoproteins, opening new opportunities for the application of flash luminescence assays in HTS. Recently, the three photoproteins by several companies such as PerkinElmer and Axxam have been commercialized. So, we selected i-photina which has the highest luminescence signal and good stability compared with two other photoproteins. Codon usage of i-Photina was optimized for both E. coli and mammalian cells. The encoding gene of i-photina was synthesized and overexpressed in E. coli. Then i-Photina was characterized and compared with aequorin. i-Photina showed about a 14-fold higher bioluminescence signal than aequorin. Ca2+ sensivity of i-Photina was found to be slightly less than aequorin. In respect to other measured properties such as degree of stability, bioluminescence spectrum and decay half-life time, these two photoproteins were almost the same. i-Photina is an improved version of photoprotein that in many ways is superior to use of other Ca2+ indicators for HTS assays and can also be considered as a potential candidate for creating genetic mutations improved the bioluminescence propertises.

Aequorin is a calcium sensitive photoprotein composed of apoaequorin (189 amino acid), oxygen and coelenterazine (imidazopyrazine chromophore). Activity of this photoprotein in calcium presence is blue photon emission (469 nm). Studies have shown that three amino acids H16, Y82, W86 are involved in color shift and activity of Aequorin. In this work, for study of structural-functional relationship, combinational mutant was used for determine of the interest places importance in single and combination state. Using site directed mutagenesis, the single and multiple mutations done in plasmid and mutated aequorin in single and multiple states were prepared. Next, thermostability, activity and color shift of produced protein determined using luminometer and spectrofluorimetry, respectively. We found that W86 and H16 play a more critical role in color shift and activity respectively. In addition, mutation of W86 provides better termostability compared to other mutations. This investigation was shown that the role of W86 in aequorin shift light and thermostability is better than Y82 and H16 and this mutant could be used for effective protein production in sensor and other applications.

In recent years, the biological methods have been considered as a most promising candidate for the cancer treatment. One of the most applicable methods is recombinant proteins. ONTAK immunotoxin is a special drug for cancer therapy of leukemia, lymphoma and melanoma. With respect to the dramatic growth of cancer in today's society, production of recombinant protein drugs seems to be necessary. In this research, optimization of complex culture medium for the expression of ONTAK recombinant has been studied. In this experimental study, various concentrations of IPTG (Isopropyl Thiogalactopyranoside) were used as an inductor and the medium components and induction time optimized for expression of ONTAK immunotoxin. The cell concentration was measured by optical density at 600 nm and the protein expression levels were analyzed by SDS-PAGE gel. The modified complex culture medium containing: glucose 6 g/l; K2HPO4 12.5 g/l; KH2PO4 2.3 g/l; Yeast Extract 20 g/l; Tryptone 10 g/l; were determined as optimal medium. OD600nm=3.0 was determined as the best time for induction by IPTG at a concentration of 0.1mM. The maximum amount of the expression of the target protein was determined at OD600nm=5.5. The adding of additive carbon source (glucose) to the complex medium was caused a better ONTAK immunotoxin expression as compared with the amount of expression observed in LB medium. The amount of produced biomass on the optimized medium increased over 3.5 times in comparison to LB medium.