Background and objectives: HIV-1 Nef and Vpr antigens have been described as suitable candidates for therapeutic HIV vaccine development. The aim of this study was to generate Nef-Vpr fusion gene construct and to clone the construct into pET-23a (+), a PROKARYOTIC EXPRESSION VECTOR. Methods: HIV-1 Nef and Vpr genes were PCR-amplified from the pNL4-3 plasmid using specific primers and Pfu DNA polymerase. Results of PCR amplification were visualized by electrophoresis on 0. 8% agarose gel. At first, the amplified Nef fragment was cloned into NheI and BamHI restriction sites of pET-23a EXPRESSION VECTOR. Next, cloning of Vpr gene was performed into BamHI and HindIII restriction sites of the pET-23a-Nef VECTOR. Finally, purity of the recombinant pET-23-Nef-Vpr construct was determined by NanoDrop spectrophotometry. Results: PCR amplification of Nef and Vpr genes was confirmed by detection of 620 bp and 291 bp bands, respectively. Cloning of the Nef-Vpr construct into the VECTOR was confirmed by detection of a 911 bp fragment following enzymatic digestion with NheI and HindIII and sequencing. Conclusion: The successful construction of recombinant fusion plasmid encoding a chimeric Nef-Vpr gene was performed in a PROKARYOTIC EXPRESSION VECTOR for development of HIV-1 recombinant protein vaccine in near future.

Tetanus is a disease caused by tetanus toxin, a potent inhibitor for the release of inhibitory neurotransmitter in the central nervous system that causes spastic paralysis. Fragment C (52 kD) of this toxin is responsible for binding to the neuronal membrane. For this reason, and also its non toxigenic and immunogenic nature, this fragment might be ideal for new vaccine development. Presently, with respect to the incidence of disease in neonates and animals and the side effects of toxoid vaccine, designing a more effective and efficient vaccine for prevention of this disease is crucial. A segment of Clostridium tetani DNA corresponding to C fragment of tetanus toxin was amplified using polymerase chain reaction. This fragment was cloned into EXPRESSION VECTOR pMalc2x, under the control of the lac promoter. EXPRESSION of this plasmid in Escherichia coli was confirmed by western blotting. In this study, the VECTOR had a strong promoter to allow high level EXPRESSION of C fragment. Based on our results it appears that this recombinant plasmid may be suitable for the production and development of recombinant vaccine and also has many other applications, such as construction diagnostic kits, production hyperimmune antiserum for serotherapy and as a vehicle for drug delivery to CNS.

Background and purpose: Highly Pathogenic Influenza A(H5N1) viruses cause vast economic losses throughout the world. They may circulate in animals and be able to spread from human to human. Therefore, launching diagnostic tests are highly essential to control the influenza infection. Studies on the amino acid sequence of the neuraminidase (NA) protein of influenza viruses revealed that NA is the most immunogenic protein in naï ve animals which can simply stimulate the humoral immune system well. Materials and methods: Influenza virus NA gene (A/Indonesia/5/2005(H5N1) was cloned into pET21a and expressed in host E. coli (BL21) strains. Then, the EXPRESSION level of NA protein was optimized for different IPTG inductor concentrations and times. Results: Findings showed the integrity of pET21a-NA construct. Emerging bands with the expected molecular weight (38KDal) on SDS-PAGE and WB analysis confirmed the successful EXPRESSION of target protein in E. coli BL21 strain. In silico analysis showed integrity of major epitopes in the structure of fused version of NA produced in this work. Conclusion: The new recombinant NA has the potential to be used directly in serological tests. It could be also used in polyclonal antibody preparation which is employed as an essential material in western blot analyses and other immunological and serological studies, such as ELISA, immunocytochemistry, and immunohistochemistry.

Peroxisomes are single membrane eukaryotic organelles that perform variable functions depending on the tissue, the developmental stage or environmental conditions. Peroxisomal matrix proteins are synthesized in the cytoplasm and targeted to peroxisomes by virtue of a peroxisomal targeting signal (PTS). One of the peroxisomal matrix proteins-Peroxisomal Protein (PEP)-has shown different pattern of EXPRESSION in mouse embryo in various tissues, but the reason is unclear. PEP-cDNA was sub-cloned in pGEX-6p-2 PROKARYOTIC EXPRESSION VECTOR in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins later. PEP-cDNA was inserted downstream of FLAG gene and then inserted in pUcD2SRMCSHyg eukaryotic EXPRESSION VECTOR to express tagged-PEP protein for transient transfection analysis and identifying localization of PEP protein. Our results clearly demonstrated that PEP and FLAG-PEP were inserted into the VECTORs appropriately and they were free from mutation.

Background and Objectives: Stem cell factor (SCF) is a 28-40 kDa glycoprotein which plays an important role for the proliferation and differentiation of hematopoietic stem cells (HSCs). SCF binds to the C-kit receptor, and improves the survival of HSCs in vitro. SCF is one of the essential supplements for cultivation of HSCs in vitro. It plays a vital role to increase the number and size of HSC colonies.Materials and Methods: In this experimental study, glycosylation of SCF does not seem to influence its biological activity; therefore, it would be possible to produce it in PROKARYOTIC EXPRESSION system. The aim of this study was isolation, cloning and EXPRESSION of SCF in the Rosetta EXPRESSION host In addition to the features of PROKARYOTIC EXPRESSION system, Rosetta was expected to provide rare codons of eukaryotic proteins and increase the recombinant protein EXPRESSION level.Results: The SCF coding sequence was isolated and amplified using the specific primers and cloned in E.coli TOP10 using pET-32a EXPRESSION VECTOR. The recombinant construct was confirmed by PCR, digestion and sequencing. The recombinant VECTOR was transformed to the EXPRESSION host, Rosetta.Conclusions: The SCF coding sequence was successfully isolated and cloned into the EXPRESSION VECTOR pET- 32a, followed by recombinant protein EXPRESSION in Rosetta host strain.

Peroxidase is an enzyme which catalyzes oxido-reduction reaction between hydrogen peroxide and reductants. It has been extensively used in biotechnology and related sciences and can be considered as the most widely used enzyme. Thereby, it is necessary to find other new sources of the enzyme due to its importance. Up to now, horseradish peroxidase (HRP) that is one of neutral isoenzymes of Armorica rusticana (Horseradish) has been purified and because of the low level of production and high cost of purification, it is not a frugal enzyme. Thus, purification of the enzyme from other plants as well as obtaining of the recombinant protein from PROKARYOTIC host can have scientific and economic benefits. For these reasons, here, the peroxidase gene from neutral isoenzymes of L. draba is studied and then cloned into the EXPRESSION VECTOR pET28a. Recombinant DNA is transformed into competent E. coli BL21 (DE3) cells. Analysis of clones is performed by two techniques: clones PCR and digestion of DNA by restriction enzyme. BL21 (DE3) cells harboring the EXPRESSION constructs are grown in LB medium and then induced with IPTG. The cell pellet is broken in buffer in the presence and absence of reducing agent and then protein EXPRESSION analyzed by SDS-PAGE. The SDS-PAGE analysis shows that the recombinant protein is successfully expressed. Also, based on the results, majority of the protein was expressed as insoluble inclusion body.

Background and Aims: Influenza A virus of Orthomyxoviridae family is able to create pandemic influenza. Vaccination is the most effective way to prevent influenza virus infection. Matrix protein 2 (M2) is a homotetramer ion channel with 97 amino acids length and highly conserved among influenza viruses and is considered for development of a universal influenza vaccine.Materials and Methods: We present here cloning and EXPRESSION of influenza A virus M2 protein as a fusion with 6-His tag in Escherichia coli BL21 strain. The gene was amplified by PCR and ligated into the PROKARYOTIC EXPRESSION VECTOR pET28a. The EXPRESSION of M2 protein was induced by IPTG and confirmed by SDS-PAGE and western blotting. The desired protein was purified with affinity chromatography on a Ni-TED resin column and has to be evaluated in animal models for further studies.Results: The results of sequencing showed that M2 gene was cloned in pET28a properly in frame to histidine tag and the product was confirmed by xpreimmune reaction of monoclonal anti-M2 antibody to recombinant M2 in western blotting.Conclusion: This study might provide a basis for production of a universal and broad-spectrum human influenza vaccine.

Chitinolytic enzymes are involved to break down chitin waste products such as chitin shells of crustaceans and cell walls of fungi and production of chitin oligosaccharides. Ideal catalytic properties of these enzymes make them an attractive target for use in industry or as a biocontrol. In this study to amplify and cloning ofchit36 gene from native Iranian isolate, Trichoderma atroviride PTCC5220, Specific primers (chit36pf/chi36r) were designed and then intended to produce in periplasmic form, amplified fragment was cloned into pET26b (+) VECTOR. The new construct was transformed intoE. coli BL21-DE3 bacteria. The results of SDS-PAGE showed no evidence of protein EXPRESSION in any of the conditions of temperature and different levels of IPTG at different cell fractions. So EXPRESSION of Chit36 protein with the removal of signal peptide (to produce cytoplasmic form) with histidine sequence at the carboxyl end was planned. Bacteria containing recombinant construct were evaluated at different induction conditions. Despite detection of any band in any induction conditions using SDS-PAGE, low EXPRESSION of proteins in two colonies were confirmed by Western blot assay. In order to optimize the EXPRESSION condition, recombinant protein EXPRESSION levels relative to the total bacterial protein in different induction conditions was evaluated using the quantitative measurements of protein bands on SDS-PAGE gels. The results showed that the best condition to induce EXPRESSION is at OD600: 0.3 and 1 mM IPTG and incubation time of 6 hours. In these conditions EXPRESSION level of recombinant protein relative to total protein was 45.57%.

The identification of a large number of antigens with potential for development of new tuberculosis vaccine has been accomplished in recent years. This study was designed for cloning and EXPRESSION of ESAT-6 as a potent antigen of Mycobacterium tuberculosis. Selected gene (Rv3875) was amplified by PCR and product was ligated into expressing plasmid VECTOR pQE30 and recombinant pQE30-ES plasmid was constructed. This hybrid VECTOR was transformed in E. coli M15 and expressed in optimal condition. The expressed protein was analyzed on SDS-PAGE and confirmed by western blotting using specific antisera to ESAT-6. We successfully cloned and expressed ESAT-6 (His)6 from M. tuberculosis H37Rv genome. As well as usage for serodiagnosis, this recombinant protein offers the potential development of other vaccine formats such as DNA or subunit vaccines against tuberculosis.