Showing papers on "Cloning published in 2019"
TL;DR: The fundamental mechanism underlying in vivo cloning is clarified and a strain is constructed that was optimized for in vivo clones by using a single microcentrifuge tube.
Abstract: Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as in vivoE. coli cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC. IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo cloning and also constructed a strain that was optimized for in vivo cloning. Additionally, we streamlined the procedure of in vivo cloning by using a single microcentrifuge tube.
45 citations
TL;DR: Given the simplified protocols and minimal requirements, cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the molecular biology community.
37 citations
TL;DR: This work describes the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site, which it is believed that this is the smallest high- copy cloning vector ever described.
Abstract: Minimal plasmids play an essential role in many intermediate steps in molecular biology. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site. We believe that this is the smallest high-copy cloning vector ever described.
15 citations
TL;DR: ABC cloning as mentioned in this paper uses a thermostable DNA polymerase to combine three overlapping DNA fragments into a recombinant vector that can be immediately transformed into competent cells and is more rapid and efficient than previously described methods.
11 citations
TL;DR: Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription and rescued some deficient RNA-editing sites in cloned two-cell embryos.
Abstract: Somatic cell nuclear transfer (SCNT) is a very powerful technique used to produce genetically identical or modified animals. However, the cloning efficiency in mammals remains low. In this study, we aimed to explore the effects of vitamin C (Vc)-treated donor cells on cloned embryos. As a result, Vc treatment relaxed the chromatin of donor cells and improved cloned embryo development. RNA sequencing was adopted to investigate the changes in the transcriptional profiles in early embryos. We found that Vc treatment increased the expression of genes involved in the cell–substrate adherens junction. Gene ontology (GO) analysis revealed that Vc treatment facilitated the activation of autophagy, which was deficient in cloned two-cell embryos. Rapamycin, an effective autophagy activator, increased the formation of cloned blastocysts (36.0% vs. 25.6%, p < 0.05). Abnormal expression of some coding genes and long non-coding RNAs in cloned embryos was restored by Vc treatment, including the zinc-finger protein 641 (ZNF641). ZNF641 compensation by means of mRNA microinjection improved the developmental potential of cloned embryos. Moreover, Vc treatment rescued some deficient RNA-editing sites in cloned two-cell embryos. Collectively, Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription. This study provided useful resources for future work to promote the reprogramming process in SCNT embryos.
11 citations
TL;DR: The development of three vectors for TA cloning and blunt-end cloning are described, including pCRT and pCRZeroT were designed to improve the efficiency of TA cloning, and a direct PCR cloning protocol with non-digested circular vectors and PCR products was developed.
Abstract: An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene ( https://www.addgene.org/ ).
10 citations
TL;DR: Results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.
Abstract: Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.
8 citations
TL;DR: A robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis is established and it is concluded that the system can reliably generate variants for genome-level engineering of algal mitochondria.
Abstract: With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria. HIGHLIGHTS TAR-cloned mitochondrial genome of P. tricornutum in yeast Developed PCR-based cloning method to create designer algal mitochondrial genomes Stably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts
8 citations
TL;DR: The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype, highlighting the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.
7 citations
7 citations
TL;DR: Cloning and expression of an optimized interferon Alpha2b in Escherichia coli strain BL21 (DE3), Ann Trop Med & Public Health; 22(IV): S376 confirmed successful expression of IFN α2b.
Abstract: Interferon alpha 2b gene (INF α2b) as a protein with antiviral and antitumor activities is potentially a valuable therapeutic proteins to work on. Prior to a large scale production of the target protein, it is recommended to examine it in an experimental scale, so that bacterial host could be a proper choice as it leads us to a deep insight of the subject. In this research, INF α2b sequence obtained from NCBI gene data bank, and after optimization it was subjected to be cloned and expressed in pET28a+. In order to primary examination of the target protein, Escherichia coli was considered as a prokaryotic expression system. IPTG induction of the protein in bacteria cells containing the construct pET: IFN, followed by resolving total proteins through SDS-PAGE. The expected size of the investigated protein, about 24kDa, observed through gel separation. Further assessment via western blotting confirmed successful expression of IFN α2b. Keywords; Recombinant protein; Bacterial expression system; IPTG induction; Western blotting How to cite this article: Alrseetmiwe DS, Almayah AA, et al (2019): Cloning and expression of an optimized interferon Alpha2b in Escherichia coli strain BL21 (DE3) , Ann Trop Med & Public Health; 22(IV): S376. DOI: http://doi.org/10.36295/ASRO.2019.221226
TL;DR: CDNA cloning of IP3-binding core (IBC) domain is presented, encoding only residues 224-604 of human IP3R type 2 that binds to IP3 with high affinity and bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.
TL;DR: In this chapter several molecular methods suitable for high-throughput cloning are reviewed and made possible the cloning of a huge number of DNA sequences in a high-Throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization.
Abstract: The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.
01 Jan 2019
TL;DR: Genomic full length sequence of HLA‐A*24:20:01:01, was identified by cloning and sequencing from a Chinese donor.
Abstract: Genomic full length sequence of HLA-A*24:20:01:01, was identified by cloning and sequencing from a Chinese donor.
01 Dec 2019
TL;DR: This review highlights the recent advances in QPM development and its practical application in the post genomic era, genomic and physical composition and evolution of zein family, and expression, regulation and downstream role ofZein genes in endosperm development.
Abstract: Maize (Zea mays) is a cereal crop of global food importance. However, the deficiency of essential amino acids, more importantly lysine, methionine and tryptophan, in the major seed storage zein proteins makes corn nutritionally of low value for human consumption. The idea of improving maize nutritional value prompted the search for maize natural mutants harboring low zein contents and higher amount of lysine. These studies resulted in the identification of more than dozens of maize opaque mutants in the previous few decades, o2 mutant being the most extensively studied one. However, the high lysine contents but soft kernel texture and chalky endosperm halted the widespread application and commercial success of maize opaque mutants, which ultimately paved the way for the development of Quality Protein Maize (QPM) by modifying the soft endosperm of o2 mutant into lysine-rich hard endosperm. The previous few decades have witnessed a marked progress in maize zein research. It includes elucidation of molecular mechanism underlying the role of different zein genes in seed endosperm development by cloning different components of zein family, exploring the general organization, function and evolution of zein family members within maize species and among other cereals, and elucidating the cis- and trans-regulatory elements modulating the regulation of different molecular players of maize seed endosperm development. The current advances in high quality reference genomes of maize lines B73 and Mo17 plus the completion of ongoing pan genome sequencing projects of more maize lines with NGS technologies are expected to revolutionize maize zein gene research in near future. This review highlights the recent advances in QPM development and its practical application in the post genomic era, genomic and physical composition and evolution of zein family, and expression, regulation and downstream role of zein genes in endosperm development. Moreover, recent genomic tools and methods developed for functional validation of maize zein genes are also discussed.
TL;DR: The first equine cloned foal born in Australia was produced with somatic cells treated with chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning.
Abstract: Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.
01 Jan 2019
TL;DR: Results of this study would help elucidate the importance of serpins in the regulation of PCD in rice and could lead to further research aimed at generating disease-resistant transgenic plants.
Abstract: Programmed Cell Death (PCD) is an important mechanism of plant immune response against diseases. Serine protease inhibitors (‘serpins’) are a conserved superfamily of proteins that inhibit serine protease targets and prevent programmed cell death (PCD) in plants, in absence of pathogen infection. In this project, putative serpin genes in rice (Oryza sativa) which are homologous to necrotic/‘Nec’ serpin genes in Drosophila are identified by bioinformatic analyses. This is followed by cloning of specific exon sequences of the rice serpin genes identified. Finally, one of the cloned sequences is utilized in a series of steps to produce interfering RNA (RNAi), to block the gene expression. Results of this study would help elucidate the importance of serpins in the regulation of PCD in rice. The work could lead to further research aimed at generating disease-resistant transgenic plants.
TL;DR: The novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
Abstract: The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method—one-Pot cloning for influenza A virus—was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
TL;DR: Using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.
Abstract: Cosmid libraries can represent an entire genome in a library of circular DNA molecules, allowing for the faithful amplification, cloning and isolation of large genomic DNA fragments. Moreover, using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.
TL;DR: DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12 and could be recommended for addition to culture media.
Abstract: Abstract Dermal fibroblasts are commonly used as donors of genetic material for somatic cell nuclear transfer in mammals. Basic fibroblast growth factor (bFGF) is a cytokine that regulates proliferation and differentiation of different cell types. The study was aimed at optimizing the cell culture protocol for cat dermal fibroblasts by assessing the influence of culture media and different doses of bFGF on proliferation of fibroblasts and their viability in terms of cell banking and somatic cloning of felids. In Experiment I, skin biopsies of domestic cats were cultured in DMEM (D) and/or DMEM/F12 (F), both supplemented with 5 ng bFGF/ml (D-5, F-5, respectively). After the primary culture reached ~80% of confluency, the cells were passaged (3–4 times) and cultured in media with (D-5, F-5) or without (D-0, F-0) bFGF. To determine the optimal doses of bFGF, in Experiment II, secondary fibroblasts were cultured in DMEM with 0 (D-0), 2.5 (D-2.5), 5 (D-5) or 10 (D-10) ng bFGF/ml. The results showed that in D-5 the cells proliferated faster than in D-0, F-5 and F-0. Due to their poor proliferation, passages IV were not performed for cells cultured in F-0. In experiment II, a dose-dependent effect of bFGF on proliferation of cat dermal fibroblasts was found. In D-5 and D-10, the cells exhibited higher (P<0.05) proliferation compared with D-0. In D-2.5 the cells showed a tendency to proliferate slower than in D-5 and D-10 and at the same faster than in D-0. In conclusion. DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12. Supplementation of culture medium with bFGF has a beneficial effect on cat dermal fibroblast proliferation and could be recommended for addition to culture media.
TL;DR: Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5'-overhangs, a fusion protein was integrated into the third intracellular loop of a G-protein-coupled receptor with nine different linker boundaries, using only a single plasmid linearization reaction.
TL;DR: A protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells is described, the procedures to establish single-cell-derived Porcine fibro Blast cell colonies carrying the desired genetic modifications are described, and the handmade cloning technique to generate cloned embryos ready for embryo transfer is described.
Abstract: Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired genetic modifications, and the handmade cloning (HMC) technique to generate cloned embryos ready for embryo transfer.
TL;DR: Analytical analyses suggest that the somatic embryogenesis does not lead to major genomic instability and clones are identical copies of mother plants, even with detected differences in the expression of genes that influence the response of in vitro cultivation.
Abstract: Our analyses suggest that no major genome organization occurred during SE process implying the nonoccurrence of somaclonal variation. However, the genetic background determines the quality of the in vitro response. Cloning of superior coffee plants by somatic embryogenesis can assist breeding programs on reducing the cost and time for launch of new cultivars. This study aimed to evaluate the efficiency of this methodology for cloning coffee trees with high heterozygosity, and to gather evidence that clonal progenies are faithful copies of mother plants. Selected plants IAC1 and IAC 2 from Coffea arabica breeding populations, resistant to leaf rust and leaf miner, respectively, were cloned via indirect somatic embryogenesis. Expression of selected genes involved in biological processes potentially affected by in vitro cultivation was evaluated by quantitative analysis. Genes encoding proteins associated with maintenance of DNA integrity and control of cell cycle presented predictable expression patterns along the clonal multiplication process. There were differences in the expression pattern of genes linked to in vitro cultivation-related stress, which were observed comparing either IAC 1 and IAC2 genotypes or clones and their corresponding mother plant. Those analyses suggest that the somatic embryogenesis does not lead to major genomic instability and clones are identical copies of mother plants, even with detected differences in the expression of genes that influence the response of in vitro cultivation.
10 Mar 2019
TL;DR: In this paper, a mouse IL-2 recombinant was produced in BL21 and pET-21b (+) expression system at 24∘C in the soluble form.
Abstract: Backgrounds and objectives: Many proteins have been expressed so far in bacterial host. Due to its simple culture conditions, having a short life cycle, and easily genetic manipulation, E.coli have been regarded as a preferable host to produce recombinant proteins, but protein cloning in bacterial host have many challenges. Therefore, we aimed to review some of these problems by an experience from mice IL-2 recombinant. Methods: cDNA synthesis was performed after RNA extraction of mouse splenocytes. PCR product purification carried out after IL-2 coding sequence amplification and was ligated into the pET-21b (+) vector and transformed into the competent BL21 E.coli. Expression and purification of recombinant mouse IL-2 were done using IPTG inducer and metal affinity chromatography respectively. Results: DNA sequencing confirmed the accuracy of the insertion process. A 23 kDa exogenous protein was observed on the SDS-PAGE. Specificity and concentration of produced mouse recombinant IL-2 protein were confirmed by western blotting and BCA methods. Conclusion: Recombinant IL-2 was produced in BL21 and pET-21b (+) expression system at 24∘C in the soluble form.
26 Jul 2019
TL;DR: In this article, a new technique that uses the system known as CRISPR/Cas9 has opened the borders globally, to the era of gene editing, in a faster, easier and more accessible way.
Abstract: Since the discovery of the double helix by Watson and Crick, biotechnological advances related to the world of genetics, have yielded rapid jumps for humanity. Major milestones such as DNA sequencing, the Human Genome Project, the cloning of the Dolly sheep, and even the development of genetically modified phosphorescent cats to shine in the dark, among others, have been possible with these advances. Genetic engineering is a possibility thanks to this development of science, and so since 2012, the emergence of a new technique that uses the system known as CRISPR/Cas9 has opened the borders globally, to the era of gene editing, in a faster, easier and more accessible way. When talking about new technologies of genetic modification, especially if it is about the modification of the germ line, the bioethical impact must be
TL;DR: LtTSA and its encoded TSA protein were found to be highly immunogenic and similar to TSA proteins previously tested as a vaccine candidate.
Abstract: Background/aim: Thiol-specific antioxidant (TSA) protein is one of the most promising molecules among candidates for vaccine against cutaneous leishmaniasis. It was found to be significantly protective against different Leishmania species. In this study, cloning and molecular characterization of thiol-specific antioxidant gene of L. tropica Turkey isolate (LtTSA) were aimed. Materials and methods: LtTSA was amplified by PCR using the specific primers of TSA gene and cloned into the pcDNA3.1 vector. The cloning was confirmed by PCR screening, restriction enzyme reactions, and DNA sequence analysis. Finally, three-dimensional structure and antigenic properties of the protein encoded by the LtTSA were determined.Results: Six hundred base pair bands belonging to LtTSA were shown with electrophoresis. It was found that LtTSA and its encoded protein have high similarity with different Leishmania species. LtTSA protein consisting of 199 amino acids was found to have 7 different antigenic regions. Conclusion: LtTSA and its encoded TSA protein were found to be highly immunogenic and similar to TSA proteins previously tested as a vaccine candidate.
TL;DR: A conserved sequence of ORFVDNA Pol was synthesized and cloned in pCDH-CMV-MCSEF1-cGFP-T2A-Puro lentiviral vector at the upstream of GFP gene and the validity of cloning was confirmed.
Abstract: Received: Revised: Accepted: Published online: June 24, 2018 August 21, 2018 September 06, 2018 March 07, 2019 Contagious ecthyma or Orf is a highly contagious; zoonotic disease and despite the application of some control strategies against it, remains prevalent worldwide. Numerous measures such as anti-viral gene therapies are frequently employed to control viral infections such as ORFV. One way to evaluate these measures is the cloning of the viral target sequence into suitable vectors for the preparation of cell lines expressing subgenomic replicons of the virus. In this study a conserved sequence of ORFVDNA Pol was synthesized and cloned in pCDH-CMV-MCSEF1-cGFP-T2A-Puro lentiviral vector at the upstream of GFP gene. The validity of cloning was confirmed by restriction enzyme digestion and sequencing. Therefore, this recombinant plasmid will be available to produce lentiviral vectors carrying this gene and after infection of Eukaryotic cells with such lentiviral vectors the expression of target gene will be induced. ©2018 PVJ. All rights reserved