• Asian-Australasian Journal of Animal Sciences
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• v.21 no.9
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• pp.1286-1295
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• 2008

Leptin is produced by adipocytes and its role in the regulation of lipid metabolism, feed intake, productive and reproductive performance of domestic animal species has been greatly stressed and extensively investigated in recent years. This study was conducted to develop a radioimmunoassay (RIA) for the estimation of plasma bovine leptin and to determine plasma leptin concentration in fattening Japanese Black cattle (Wagyu) and its crossbreds at commercial farms. Relationships of plasma leptin with plasma vitamin A and age of crossbred cattle were also determined. Recombinant bovine leptin (rbleptin) was produced by the E. coli overexpressed leptin as a GST (glutathione S-transferase)-fusion protein. Then antiserum against bovine leptin was obtained by its immunization in rabbits. Using this antiserum, a bovine specific RIA was developed and plasma leptin level was determined in 120 crossbred fattening cattle (WagyuHolstein, 50:50) at commercial farms. The plasma leptin level increased with the age of cattle and its level was greater in the crossbred heifers than in the steers. Plasma vitamin A level was negatively correlated with plasma leptin level in crossbred heifers and steers. This relationship was stronger in heifers than in steers. Plasma leptin was gradually increased with advancing age in fattening Wagyu cattle. In conclusion, development of a bovine specific RIA to estimate plasma leptin will contribute to better understanding of the role of leptin in cattle.

• Biomedical Science Letters
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• v.19 no.4
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• pp.344-347
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• 2013

The S100A8 and S100A9 proteins play important roles in inflammatory diseases. The house dust mite acts as a major allergen that induces allergic diseases. We investigated the effect of the house dust mite on S100A8 and S100A9 protein expression in monocytes. We also examined the effect of CCL2, a powerful monocyte chemoattractant, on the expression of both proteins. Extract of Dermatophagoides pteronissinus (DP), recombinant Der p 1 and Der p 2, or CCL2 had no effect on S100A8 and S100A9 expression in human monocytic THP-1 cells. Monocytes were isolated from healthy donors and treated with DP, Der p 1, and Der p 2. S100A8 expression in monocytes increased after a 24 h stimulation with DP, Der p 1, and Der p 2, and CCL2 also increased S100A8 production. However, S100A9 expression in monocytes was not altered by DP, Der p 1, Der p 2, or CCL2. These results indicate that house dust mite and CCL2 may trigger an inflammatory response by altering S100A8 expression.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1471-1480
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• 2010

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.35-40
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• 2004

Rotavirus virus-like particle (VLP) composed of VP2, VP6, and VP7 was expressed in the Baculovirus Expression Vector System (BEVS). Sf9 cell, a host of the baculovirus, was cultured from a 0.5-1 spinner flask to the 50-1 bioreactor system. Sf9 cell was maintained at cell density between 3.0E＋05 and 3.0E＋06 cells/ml and grew up to 1.12E＋07 cells/ml in the bioreactor. Growth kinetics was compared under different culture systems and showed similar growth kinetics with 20.1-25.2 h of doubling time. Early exponentially growing cell culture was infected with three recombinant baculoviruses expressing VP2, VP6, and VP7 protein at 1.0, 2.0, and 0.2 moi, respectively. The expression of rotavirus proteins was confirmed by Western blot analysis and its three-layered virus-like structure was observed under an electron microscope. Rotavirus VLP was semipurified and immunized in ICR mice intramuscularly. Rotavirus-specific serum antibody was detected from 2 weeks after the immunization and lasted at least 21 weeks of the post-immunization, indicating its possible use as a vaccine candidate.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.1-7
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• 1997

Recently the production of transgenic animals to express foreign proteins in mammary glands has been a routine procedure. However, it still takes a considerable time and effort, and is faced with various technical challenges until the protein of interest is successfully made. Thus, a development of an a vitro model for mamm a ary-specific gene expression for recombinant genes was carried out in this study. To this end, bovine $\alpha$$_S1$ casein cDNA was inserted at the multiple cloning site of pMSG vector under the control of MMTV promoter. MCF$_7$ cells were tran sfected with pMSG $\alpha$$_S1$ CN by CaP0$_4$ precipitation. Transfectants were selected in HAT medium and induced with dexamethasone. The cells were analyzed with chicken anti-casein and FITC-labeled rabbit anti-chicken antibodies. The results showed that dexamethasone induced 30-40 fold increase in the MMTV- $\alpha$$_S1$ casein e expression. Therefore MCF$_7$ cells, which have multiple steroid receptors, along with pMSG vector can be used as an in vitro model for the study of mammary-specific gene expression.

• Journal of Microbiology
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• v.44 no.3
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• pp.293-300
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• 2006

The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL2l (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GMI-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92 % purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.603-609
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• 2002

DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.316-325
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• 2012

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.271-276
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• 2017

A highly thermostable ${\beta}-(1-4)-glucanase$ (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ${\beta}-{\small{D}}-glucan$ from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at $90^{\circ}C$ and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at $85^{\circ}C$. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

• The Plant Pathology Journal
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• v.36 no.6
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• pp.591-599
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• 2020

Phytophthora root and stem rot reduce soybean yields worldwide. The use of R-gene type resistance is currently crucial for protecting soybean production. The present study aimed to identify the genomic location of a gene conferring resistance to Phytophthora sojae isolate 2457 in the recombinant inbred line population developed by a cross of Daepung × Daewon. Singlemarker analysis identified 20 single nucleotide polymorphisms associated with resistance to the P. sojae isolate 2457, which explained ~67% of phenotypic variance. Daewon contributed a resistance allele for the locus. This region is a well-known location for Rps1 and Rps7. The present study is the first, however, to identify an Rps gene locus from a major soybean variety cultivated in South Korea. Linkage analysis also identified a 573 kb region on chromosome 3 with high significance (logarithm of odds = 13.7). This genomic region was not further narrowed down due to lack of recombinants within the interval. Based on the latest soybean genome, ten leucine-rich repeat coding genes and four serine/ threonine protein kinase-coding genes are annotated in this region, which all are well-known types of genes for conferring disease resistance in crops. These genes would be candidates for molecular characterization of the resistance in further studies. The identified R-gene locus would be useful in developing P. sojae resistant varieties in the future. The results of the present study provide foundational knowledge for researchers who are interested in soybean-P. sojae interaction.