• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.81-88
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• 2002

The purpose of this study was to conduct diagnosis of bovine paratuberculosis in Kangwon area. Blood samples were collected from 2,261 dairy cows of 162 herds, and the ELISA and immunoblotting using recombinant 34KDa protein of M. paratuberculosis were conducted. The feces collected from dairy cows were cultured on HEY medium with mycobactin-J and PCR was conducted with washing solution of medium 4 weeks after culture. The ELISA had sensitivity of 83.3% and specificity of 96.7%. And the immunoblotting had sensitivity of 83.3% and specificity of 100%. Of the 2,261 dairy cows, 371 cows(16.4%) were positive in ELISA and 75 cows(3.3%) were positive in immunoblotting. And of the 162 herds, 109 herds(67.3%) had an apparent paratuberculosis prevalence by ELISA and 40 herds(24.7%) by immunoblotting. The geographic distribution of herds with paratuberculosis was not uniform. Of the 241 feces samples including 110 feces from ELISA positive cow, 9 feces were positive in culture and PCR. PCR was able to detect the growth of M. paratuberculosis as early as 4 weeks of culture.

• Korean Journal of Veterinary Service
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• v.34 no.1
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• pp.5-12
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• 2011

Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is a highly contagious viral disease in chicken. It causes heavy economic loss by immune suppression and high mortality. The IBDV, designated Avibirnavirus in the Family Birnaviridae, has a double-stranded RNA genome formed by two segments, segment A and segment B. Segment A encodes a 108 KDa polypeptide that is self-cleaved to produce pVP2, VP3 and VP4, and later pVP2 is cleaved to VP2. The VP2 contains the antigenic regions responsible for elicitation of neutralizing antibodies and VP3 is a major immunogenic protein of IBDV. In this study, monoclonal antibodies (MAbs) specific for IBDV were produced and characterized. All 15 MAbs were specific for IBDV and did not react with other viruses used in this study. The protein specificity of MAbs was determined by comparing the reactivity patterns of each MAb with IBDV VP2 and VP234 recombinant baculoviruses and Western blot analysis. As a result, 7 MAbs (1F5, 2C8, 2F4, 3C7, 4C3, 6F11, 6G5) and 5 MAbs (2A4, 2G2, 3F5, 3G2, 4F10) were specific for VP2 and VP3, respectively. The protein specificity of 3 MAbs (2B8, 3F7, 3F8) were not determined. Five (2C8, 2F4, 4C3, 6F11, 6G5) of the VP2-specific MAbs had a neutralizing activity against IBDV. Some MAbs reacted with IBDV-infected bursa of Fabricius by indirect fluorescence antibody (IFA) and immunohistochemistry (IHC) assay. The MAbs produced in this study would be used for diagnostic reagents for the detection of IBDV infection.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.224-224
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• 2017

In plants, cysteine(Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur containing secondary products. Cys formation is involved in the consecutive two reactions using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast and mitochondria. OASTL is able to produce mimosine with 3-hydroxy-4-pyridone (3H4P) in lieu of $H_2S$ for Cys. In this report, we describe the first time cloning, purification and characterization of cytosolic(cy)OASTL from M. pudica and its expression in Escherichia coli and try to find out the cross link between this OASTL and the mimosine formation and to elucidate the metabolic role of cy-OASTL in M. pudica. The purified recombinant protein was 34.7 KDa. The optimum reaction pH and temperature was 6.5 and $50^{\circ}C$, respectively. The Michaelis constant (Km) and the Vmax value of the enzyme was $252{\pm}25{\mu}M$ and $57{\pm}3{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for sulfide and $159{\pm}21{\mu}M$ and $58{\pm}2.4{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for OAS subsequently. After cleaving the His-tag, we tried to observe cy-OASTL to form mimosine with appropriate substrate but it was not successful. It may be concluded that cy-OASTL of the present study is only Cys specific, not mimosine.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.