• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2060-2069
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• 2017

Newcastle disease is a serious infectious disease in the poultry industry. The commercial vaccines can only offer limited protection and some of them are expensive and need adjuvants. At present, DNA vaccines are widely used. However, the immune responses induced by DNA vaccines are too slow and low. Here, we constructed the transfer vectors with a different number of C3d as molecular adjuvants (n = 1, 2, 4, or 6), and the vectors were cloned into the optimal eukaryotic expression plasmid (pVAXI-optiF) that expressed the F gene of Newcastle disease virus (NDV), and named pVAXI-F(o)-C3d1, pVAXI -F(o)-C3d2, pVAXI-F(o)-C3d4, and pVAXI-F(o)-C3d6, respectively. Cell transfection test indicated that pVAXI-F(o)-C3d6 showed the highest expression. In vivo immunization showed that the chickens immunized with pVAXI-F(o)-C3d6 intramuscularly induced better immune responses than the chickens immunized with the other plasmids. The protective efficacy of pVAXI-F(o)-C3d6 was 80% after challenge with the highly virulent NDV strain F48E9. The results in this study showed that C3d6 could be used as a molecular adjuvant to quickly induce an effective immune response to control NDV.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.138.2-139
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• 2003

Grapevine leafroll-associated 1 virus (GLRaV-1) and Grapevine leafroll-associated 3 virus (GLRaV-3), member of the genus Ampelovirus, are important viral disease of grapevine in the world. these viruses transmitted only dicotyledonous host by vectors such as mealybugs and there is no suitable herbaceous host for virus. The diseased leaves turn yellowish or reddish depending on cultivars and viruses. Viruses are existed at low concentration and ununiformly distribution in grapevine. Using small-scale double-stranded RNA (dsRNA) extraction method, reverse transcription and polymerase chain reaction (RT-PCR) product of 1Kb long which encoded of coat protein (CP) gene for both viruses was successfully amplified with a specific primers. The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined from selected recombinant cDNA clones. Sequence analysis revealed that the CP of GLRaV-1 consisted of 969 nucleotide, which encoded 323 amino acid residues and CP of GLRaV-3 consisted of 942 nucleotide, which encoded 314 amino acid residues. The CP of GLRaV-1 and GLRaV-3 has 93.8％ and 98.7％ amino acid sequence identities, respectively.

• Korean Journal of Veterinary Research
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• v.29 no.3
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• pp.407-416
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• 1989

The prospect of live vaccines consisting of genetically modified vaccinia virus expressing foreign genes is exciting, but important issues concerning safety and efficacy need to resolved. Vaccinia virus (VV) is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. The WHO smallpox eradication program, involving the extensive use of VV vaccines, resulted in the late 1970s in the elimination of one of the world's most feared diseases. This achievement is a triumph for preventive medicine and for international collaboration in public health. In 1980, WHO recommended that the routine use of smallpox vaccine should be stopped. Against this background, the prospect of li ve vaccines consisting of genetically modified VV expressing foreign antigens arising from the work of Moss, and Paoletti and their colleagues in 1982 has been greeted with enthusiasm. These investigators have shown that genes coding for immunogenic proteins can be inserted into VV DNA without impairing the ability of the virus to grow in cell culture. Moreover experimental animals infected with VV recombinants containing genes coding for a variety of immunizing proteins have been shown to be protected against challenge infection with the corresponding infectious agent. In this communication, I describe current progress in the construction of a novel plasmid vector that facilitate the insertion and expression of foreign genes in VV as well as the selection of recombinants.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.31-36
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• 1994

From the pSKH201 plasmid which had been previously cloned in our laboratory, a 3.0kbp insert DNA encoding the alkaline phosphatase of Kluyveromyces fragilis was cleaved with several restriction endonucleases and ligated int the appropriate sites of M13mp18/19 vectors and sequenced by Sanger's dideoxy chain termination method. The sequence contained a 1,638 bp open reading frame(ORP) whose similarities in nucleotide, when compared with those of Saccharomyces cerevisiae and Escherichia coli by GENETYX program, were found to be 61% and 46%, respectively. The deduced amino acid sequence consists of 546 amino acids and contains several homologous regions in the alkaline phosphatases of E. coli, S.cerevisiae and human placenta.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.375-382
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• 2010

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei, contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers, and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein secretion in L. casei.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.379-385
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• 2000

tRNA and 7SL RNA based antisense vehicles were prepared by inserting conserved anti-viral and anti-viroid domains. Anti-PVS coat protein leader sequence (ACPL) and antistructural antihairpin domain of PSTVd (AHII) were inserted in tRNA cassette; anti- zing finger domain of PVS, AHII and anti hop latent viroid ribozyme were inserted in 7SL RNA gene isolated from A. thaliana. These constructs were shown to be transcribed both, in in vitro and in in vivo conditions. However, it followed from our work that closely linked position of PoIII reference genes and PoIIII antisense genes within T-DNA lead to the impairment of RNA expression in transgenic plants. To assay in vivo transcription of antisense genes, hairy root potato cultures were established using h. tumefaciens A4-24 bearing both, Ri plasmid and PoIII-promoterless plant expression vectors with antisense RNA genes. Expression of antisense RNA in transgenic potato tissues was proven by specific RT-PCR reactions.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.229-234
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• 2006

We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.

• Journal of Life Science
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• v.10 no.4
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

• Molecules and Cells
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• v.39 no.9
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• pp.687-691
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• 2016

Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.