• Korean Journal of Microbiology
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• v.31 no.2
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• pp.136-140
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• 1993

The A. nidulans expression vector which contained trpC marker gene from A. nidulans was constructed to produce glucoamy]ase. The recombinant plasmid was introduced into auxotrophic mutant A. nidulans B17. Southern blot analysis of the genomic DNA from transformant showed that pKHG2 DNA had integrated into the A. nidulans chromosomes. Northern analysis of the total RNA from transform ant showed that mRNA of glucoamylase gene was synthesized in induction condition. Specific activity of glucoamylase was increased in transform ants. G]ucoamylase was shown to be active in non-denaturing acrylamide gel.

• Journal of Life Science
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• v.27 no.5
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• pp.524-529
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• 2017

This study was aimed to produce a transgenic zebrafish expressing the human KCNE1 gene. Initially, the entire CDS of the human KCNE1 gene was amplified from a human genomic DNA sample by polymerase chain reaction using a primer set engineered with restriction enzyme sites (EcoRI, BamHI) at the 5' end of each primer. The resultant 402 bp KCNE1 amplicon flanked by EcoR1 and BamH1 was obtained and subsequently cloned into a plasmid vector pPB-CMVp-EF1-GreenPuro. The integrity of the cloned CDS sequence was confirmed by DNA sequencing analysis. Next, the recombinant vector containing the human KCNE1 (pPB-CMVp-hKCNE1-EF1-GreenPuro) was introduced into fertilized eggs of zebrafish by microinjection. Successful expression of the recombinant vector in the eggs was confirmed by the expression of the fluorescence protein encoded in the vector. Finally, in order to assure that the stable expression of the human KCNE1 gene occurred in the transgenic animal, RNAs were extracted from the animal and the presence of KCNE1 transcripts was confirmed by RT-PCT as well as DNA sequencing analysis. The study provides a methodology to construct a useful transgenic animal model applicable to the development of diagnostic technologies for gene therapy of LQTS (Long QT Syndrome) as well as tools for cloning of useful genes in fish.

• YAKHAK HOEJI
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• v.49 no.3
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• Journal of Aquaculture
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• v.9 no.3
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• pp.293-300
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• 1996

Validities of several gene transfer methods including microinjection, electroporation and lipo-fection with luciferase gene (pRSVL), and effectiveness of mud loach expression vector which contains ARS from mud loach on production of transgenic mud loach were evaluated. Microiniection revealed the $0\~8\%$ of transgene incidence in 2-week-old fish with significant mosaicism. Electroporation and lipofection of mud loach sperm also successfully introduced the transgene into sperm cells, and transferred the foreign DNA into zygote. Gene transfer by electroporation and lipofection showed a range of $0\~28\%$ and $0\~48.1\%$ of transgene incidence, respectively in newly hatched larvae, altough most DNA introduced were gradually degraded with the development of fish. Microinjections of mud loach expression vector caused a significantly reduced survival rate of mud loach embryos with severe teratogenic effects, and ARS/Luc transgene could not be detected in normally developed fish after microinjection.

• Journal of Korea Society of Industrial Information Systems
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• v.7 no.3
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• pp.23-31
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• 2002

In this paper we propose a new feature vector for recognition of the facial expression based on Gibbs distributions which are well suited for representing the spatial continuity. The extracted feature vectors are invariant under translation rotation, and scale of an facial expression imege. The Algorithm for recognition of a facial expression contains two parts： the extraction of feature vector and the recognition process. The extraction of feature vector are comprised of modified 2-D conditional moments based on estimated Gibbs distribution for an facial image. In the facial expression recognition phase, we use discrete left-right HMM which is widely used in pattern recognition. In order to evaluate the performance of the proposed scheme, experiments for recognition of four universal expression (anger, fear, happiness, surprise) was conducted with facial image sequences on Workstation. Experiment results reveal that the proposed scheme has high recognition rate over 95%.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.49-55
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• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.301-305
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• 2001

The co-expression of the arg U gene in a double-vector expression system of recombi-nant Escherichia coli BL22(DE3)[pET-IEN2a+pAC-argU] significantly enhanced the production level of reconminant human interferon -$\alpha$2a(rhIFN-$\alpha$2a) in high cell density cultures, compared to a recombinant E. coli culture containing only the single expression vector, pET-IEN2a. The dry cell mass concentration increased to almost 100 g/L, and more than 4 g/L of rhIFN-$\alpha$2a was accumu-lated in the culture broth. Evidently, the synthesis of rhIFN-$\alpha$2a was strongly dependent on the pre-induction growtih rate and more efficient at a higher specific growth rate. The additional sup-ply of tRN $A^{Arg(AGG/AGA)}$ enhanced the expression level of the rhIFN-$\alpha$2a gene in the early stage of the post-induction phase, yet thereafter the specific production rate of rhIFN-$\alpha$2a rapidly de-creased due to severe segregational instability of plasmid vector pET-IEN2a. It would appear that the plasmid instability with only occurred to pET-IEN2a in the double vector system, was re-lated to the effect of translational stress due to the over expression of rhIFN-$\alpha$2a.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.