جستجوی مقالات مرتبط با کلیدواژه « Codon optimization » در نشریات گروه « پزشکی »

The mRNA vaccines replace our conventional vaccines (live-attenuated and inactivated vaccines) due to their high safety, efficacy, potency and low cost for their manufacturing. Since these many years the usage of these mRNA vaccines have been restricted as there are unstable and their low efficiency in in-vivo delivery. But now, these problems have been solved by recent technological advances. Many studies conducted in animal models and humans demonstrated the good results for the mRNA vaccines. This Review provides you a detailed overview of mRNA viral vaccines and considers the current perspectives and future prospects.

Production of the recombinant proteins in mammalian cells is an important issue with a bio-therapeutic purpose. Numerous efforts have been focused on the improvement of the yields of recombinant proteins, which include optimization of conventional biological processes, selection of appropriate signal peptides, codon optimization, and re-engineering of cells to produce more proteins. Stem cell factor (SCF) is a blood cytokine which activates the c-Kit receptor. This factor is crucial not only for the differentiation of hematopoietic progenitor cells but also for the survival, proliferation, and differentiation of mast cells. Recently, its therapeutic role in several diseases such as Alzheimer’s and myocardial infarction has been investigated. Therefore, the aim of this study was to design a secretory recombinant human SCF with the maximal yield in an appropriate mammalian host cell as Chinese hamster ovary (CHO) cells using the computational studies.

As the first step, computational simulation studies were carried out to design the appropriate signal peptide for the human SCF protein. Codon optimized coding sequence of hSCF was transferred into a eukaryotic expression vector (pBudCE4.1). Recombinant vector (pBudCE4.1/SCF) was transfected into CHO cells and the stably transformed cells were screened and isolated. Subsequently, the expression of SCF was defined by reverse transcription polymerase chain reaction (RT-qPCR) in stably transformed cells.

Our bioinformatics studies indicated that Azurocidin signal peptide could be a suitable signal peptide for the production of SCF proteins in the CHO cells. Accordingly, computational studies revealed that the presence of 6×His-tag did not have a significant impact on the three-dimensional structure of the protein. Furthermore, the expression of hSCF was significant in the stable CHO cells.

The use of this approach may, therefore, lead to the production of highly efficient recombinant hSCF, which would be feasible for the mass production of this factor for therapeutic purposes.

Strategy for improving the production of biopharmaceutical protein continues to develop due to increasing market demand. Human granulocyte colony stimulating factor (hGCSF) is one of biopharmaceutical proteins that has many applications, and easily produced in Escherichia coli expression system. Previous studies reported that codon usage, rare codon, mRNA folding and GC-content at 5’-terminal end were crucial for protein production in E. coli. In the present study, the effect of reducing the GC-content and increasing the mRNA folding free energy at the 5’-terminal end on the expression level of hG-CSF proteins was investigated.

Synonymous codon substitutions were performed to generate mutant variants of open reading frame (ORF) with lower GC-content at 5’-terminal ends. Oligoanalyzer tool was used to calculate the GC content of eight codons sequence after ATG. Whereas, mRNA folding free energy was predicted using KineFold and RNAfold tools. The template DNA was amplified using three variant forward primers and one same reverse primer. Those DNA fragments were individually cloned into pJexpress414 expression vector and were confirmed using restriction and DNA sequencing analyses. The confirmed constructs were transformed into E. coli NiCo21(DE3) host cells and the recombinant protein was expressed using IPTG-induction. Total protein obtained were characterized using SDS-PAGE, Western blot and ImageJ software analyses.

The result showed that the mutant variant with lower GC-content and higher mRNA folding free energy near the translation initiation region (TIR) could produce a higher amount of hG-CSF proteins compared to the original gene sequence.

This study emphasized the important role of the nucleotide composition immediately downstream the start codon to achieve high-yield protein product on heterologous expression in E. coli.

Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN-β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon.

The nucleotide sequence of the codon-optimized rhIFN-β was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-β by codon-optimized and wild-type genes were quantified using ELISA test.

The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-β by codon-optimized sequence.

These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-β. 

Helicobacter pylori (H. pylori) infection has remained as a global health problem. Animal studies demonstrated the role of H. pylori oipA gene in the development of gastric cancer. The aim of this study was the cloning and expression of Helicobacter pylori oipA gene in a bicistronic vector harboring mice IL-18 gene.

The target gene encoding oipA was amplified from a codonoptimized clone by PCR, and then double-digested by restriction enzymes. The pIRESIgk/mIL18/Fc plasmid was simultaneously digested by BstXI/NotI enzymes to elicit the eGFP segment. PCR product of oipA was inserted into pIRES-Igk/mIL18/Fc plasmid using T4 ligase. Transformation into DH5α strain was done. Cloning was confirmed by PCR, enzymatic digestion and sequencing. Expression of the oipA and IL-18 mRNA was assessed by means of TaqMan Real-time PCR.

Electrophoresis of PCR product, enzymatic digestion and sequencing showed that the H. pylori oipA gene was successfully cloned into pIRES-Igk/mIL18/Fc to generate mIL18-pIRES2-oipA plasmid. The results of Real-time PCR confirmed the successful expression of both oipA and IL-18 in mouse macrophage cell line.

Considering the role of oipA in pathogenesis of H. pylori and potent activity of IL-18 as a molecular adjuvant, the results of the present study showed that the expression of codon-optimized oipA gene in bicistronic vector including mouse IL-18 is successful. So, it could be considered as an appropriate genetic vaccine candidate for H. pylori in future investigations.

Human leukemia inhibitory factor (hLIF) is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS) treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor.
In this study we want to purify recombinant human leukemia inhibitory factor in single simple method.
This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG) used for induction. Single step immobilized metal affinity chromatography (IMAC) used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P 0.05 that is significant for our work.
Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines.
We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.