فهرست مطالب zahra aminibayat

The method of choice for prevention and treatment of infection with Human Papillomavirus (HPV) and consequently cervical cancer is the application of prophylactic and therapeutic HPV-vaccine. The present study aimed to clone the most antigenic epitopes of the E6 antigen and to express it in E. coli at the lab-scale. The sequence of immune-bioinformatically determined epitopes of E6 was synthesized in the pGH vector. The new E6 gene was cloned into the pET28 vector by double-digestion of vector and target with NcoI and XhoI restriction enzymes. The recombinant vector was transformed into DH5α and cloned E6 gene was confirmed by colony PCR and DNA sequencing. pET28 was then extracted from DH5α and transferred into the BL21(DE3) expression host. Expression optimization was performed using various parameters. Cloning was confirmed by colony PCR and sequencing and optimized expression was performed at 25°c, IPTG=0.1 mM, OD600=1. Due to the protein production in the form of inclusion bodies and unavailability of His-Tags, the recovered protein didn’t confirm by western blot and didn’t purify by Ni-NAT affinity chromatography. This study aimed to express multi-epitope recombinant protein composed of selective E6 protein epitopes in the E. coli prokaryotic expression system to achieve an effective vaccine against HPV. The produced protein might be used as a therapeutic vaccine or as a platform for HPV detection.

The method of choice for prevention and treatment of infection with Human Papillomavirus (HPV) and consequently cervical cancer is the application of prophylactic and therapeutic HPV-vaccine. The present study aimed to clone the most antigenic epitopes of the E6 antigen and to express it in E. coli at the lab-scale. The sequence of immune-bioinformatically determined epitopes of E6 was synthesized in the pGH vector. The new E6 gene was cloned into the pET28 vector by double-digestion of vector and target with NcoI and XhoI restriction enzymes. The recombinant vector was transformed into DH5α and cloned E6 gene was confirmed by colony PCR and DNA sequencing. pET28 was then extracted from DH5α and transferred into the BL21(DE3) expression host. Expression optimization was performed using various parameters. Cloning was confirmed by colony PCR and sequencing and optimized expression was performed at 25°c, IPTG=0.1 mM, OD600=1. Due to the protein production in the form of inclusion bodies and unavailability of His-Tags, the recovered protein didn’t confirm by western blot and didn’t purify by Ni-NAT affinity chromatography. This study aimed to express multi-epitope recombinant protein composed of selective E6 protein epitopes in the E. coli prokaryotic expression system to achieve an effective vaccine against HPV. The produced protein might be used as a therapeutic vaccine or as a platform for HPV detection.

Cervical cancer is the leading cause of morbidity and cancer mortality in women throughout the world and persistent infection with human papillomavirus (HPV) is the main etiology for the development of this cancer. Peptide vaccines derived from E6 and E7 oncogenes are promising candidates for HPV vaccine production due to their safety, high stability, and ease of production. In the current study, expression strains such as Escherichia coli BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS were applied for poly epitopic E6 expression and the effects of temperature, induction time, and inducer concentration on the expression level of recombinant HPV16-E6 polypeptide protein were examined in the mentioned strains.

In the present study, the optimized gene of E6 recombinant protein was cloned into pET28a vector under the control of T7 promoter and the resulting plasmid was successfully transformed into Escherichia coli strains BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS. Then, the ability of these strains to express the desired recombinant protein in different conditions was compared.  Two temperatures of 25 ° C and 37 ° C, IPTG concentrations between 0.1 and 1.0 mM, and different incubation times were selected to examine the expression level of recombinant E6 protein in these strains.

The highest level of expression was obtained in Escherichia coli BL21 (DE3) -AI strain inducing at Optical Density (OD) of 0.9, 0.1 mM IPTG, and 16 hours induction at 25 °C.

The results of this study can be used to produce E6 protein as a vaccine candidate in the treatment of HPV-related cancers.

Cervical cancer is one of the leading causes of death worldwide, causing approximately 500,000 new cases and 250,000 cancer deaths each year. Persistent infection with high-risk human papillomaviruses (HPVs), particularly type 16, is the primary cause of cervical cancer development and maintenance among women worldwide. The E6 therapeutic vaccines can induce strong anti-tumor T cell-mediated immune responses, such as cytotoxic T lymphocytes, that play vital roles in current therapeutic vaccine development. In our study, bioinformatics approaches and in silico analyzes, such as protein sequence retrieval, identification of conserved regions, drawing of pedigrees, prediction of T-cell epitopes, calculation of population coverage of predicted epitopes, and molecular docking, were used to predict the major histocompatibility complex (MHC) Class I and Class II T cell epitopes of HPV16 E6. Taking into account the scores from different steps, six CD8+ T cells and three CD4+ epitopes were selected. The fusion of the selected epitopes created a universal potential vaccine with a population coverage of 86.41%. The population coverage was obtained by evaluating the potential of these epitopes to elicit innate and acquired immunity. These theoretically confirmed peptides could be employed in a poly-epitope construct as a candidate vaccine for further analyses. Also, these results provide new insights into therapeutic vaccine development.

Creatinine amidohydrolase(EC 3.5.2.10) catalyzes the reversible conversion of creatinine to creatine. Creatininase in combination with other enzymes is used for detection of creatinine in serum and urine which is of significant value for detection of renal, muscular and thyroid functions. The aim of this study was to produce recombinant creatininase enzyme in E.coli expression system to use it in creatinine assay kit. The pseudomonas pseudoalkaligene KF707 creatininase gene has been optimized and synthesized already. Subsequently, it has been subcloned into the pET28 expression vector then the expression vector has been transformed into the BL21 (DE3) cell and induced by IPTG, afterwards the expression has been evaluated using SDS-PAGE and western blot. The recombinant protein has been purified by Ni-NTA agarose resins and enzyme activity has been analyzed. A sharp 29kDa protein band has been observed on SDS-PAGE and confirmed by western blot. More than 40% of E.coli total protein was recombinant creatinase, The recombinant enzyme was purified with approximately 100% yield. The enzyme activity analysis showed that recombinant enzyme has 14 unit/ml activity. Recombinant p.pseudoalkaligene KF707 creatininase was produced for the first time and its good production yield confirmed that E.coli was an efficient expression system for its production.

Chlamydomonas reinhardtii is a novel recombinant eukaryotic expression system with many advantages including fast growth rate, rapid scalability, absence of human pathogens and the ability to fold and assemble complex proteins accurately, however, obstacle relatively low expression level necessitates optimizing foreign gene expression in this system. The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a substantial reporter molecule for monitoring gene expression and protein localization.The fluorescence of GFP requires only UV or blue light, and, therefore, the in vivo observation of GFP expression is easy with no need for complex and costly apparatus.
In this study the codon optimized GFP reporter gene was cloned into the pChlamy3 vector using Kpn1 and Not1 restriction sites. The recombinant pChlamy3-GFP expression vector was transformed into E. coli Top10 cells. The presence of the expression cassette was verified by double digestion. Then the recombinant vector was transformed into the nucleus of C. reinhardtii for expression. Afterwards, transformed cells were analyzed by fluorescence spectroscopy using a microplate reader.
The results of fluorescence spectroscopy revealed a 28-fold more fluorescence compared to wild type in one of the samples, and this is much more than the reported results by previous studies.
It is suggested that the expression system optimized by this study can potentially be used for the production of important therapeutic proteins and other heterologous proteins in C. reinhardtii.

Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone (1-34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hPTH.
Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to Escherichia coli (E. coli) β-galactosidase (LacZ) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH (1-34) gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in E. coli (DH5α) cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 mM IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase.
By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting.
In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial β-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point (pI). This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue.