• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.6-14
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• 2022

Brucella spp. are facultative intracellular pathogens that invade, survive and proliferate in numerous phagocytic and non-phagocytic cell types, thereby leading to human and animal brucellosis. Outer membrane proteins (Omps) are major immunogenic and protective antigens that are implicated in Brucella virulence. A strain deleted of the omp16 gene has not been obtained which suggests that the Omp16 protein is vital for Brucella survival. Nevertheless, we previously constructed an omp16 conditional deletion strain of Brucella, ∆Omp16. Here, the virulence and immune response elicted by this strain were assessed in a mouse model of infection. Splenomegaly was significantly reduced at two weeks post-infection in ∆Omp16-infected mice compared to infection with the parental strain. The bacterial load in the spleen also was significantly decreased at this post-infection time point in ∆Omp16-infected mice. Histopathological changes in the spleen were observed via hematoxylin-eosin staining and microscopic examination which showed that infection with the ∆Omp16 strain alleviated spleen histopathological alterations compared to mice infected with the parental strain. Moreover, the levels of humoral and cellular immunity were similar in both ∆Omp16-infected mice and parental strain-infected mice. The results overall show that the virulence of ∆Omp16 is attenuated markedly, but that the immune responses mediated by the deletion and parental strains in mice are indistinguishable. The data provide important insights that illuminate the pathogenic strategies adopted by Brucella.

• Journal of Pharmaceutical Investigation
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• 제25권3호
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• pp.227-237
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• 1995

Omeprazole(OMP) complexes such as inclusion complexes of OMP with $hydroxypropyl-{\beta}-cyclodextrin$(HPCD) and ${\beta}-cyclodextrin({\beta}-CD)$, OMP-cholestyramine(CHL) and OMP-ethylenediamine(OMP-ED) were prepared, respectively. The partition coefficients in Witepsol H-15 /pH 7.4 phosphate buffer solution of OMP complexes$(OMP-HPCD;\;3.69{\pm}0.26,\;OMP-{\beta}-CD;\;4.08{\pm}0.21,\;OMP-CHL;\;4.36{\pm}0.25\;and\;omeprazole\;sodium(OMP-Na);\;3.64{\pm}0.37)$ were higher than that of OMP $(2.66{\pm}0.47)$. OMP was not completely dissolved until even 3 hrs, but all the OMP complexes studied were released about 100% in 20 min. The rectal suppositories containing OMP or each above OMP complex were prepared using Witepsol H-15 base, and their dissolution and stability were examined, and pharmacokinetic study were investigated after their rectal administrations to the rabbits. While the suppository containing OMP was released only less than 60% in 150 min, $OMP-{\beta}-CD$, OMP-CHL, OMP-Na and OMP-ED suppositories were all released about 65% in 20 min. Especially, OMP-HPCD suppository released OMP about 70% in 10 min. All the additives such as sodium laurylsulfate, eglumine, arginine and PVP increased drug release from OMP-HPCD suppository to some extent. The decomposition rate constants of OMP in the suppositories were $9.117{\times}10^{-3}\;day^{-l}$ for OMP suppository, $2.121{\times}10^{-2}$ for OMP-HPCD, $1.607{\times}10^{-2}$ for $OMP-{\beta}-CD$, $9.26{\times}10^{-3}$ for OMP-Na, $6.769{\times}10^{-3}$ for OMP-CHL and $5.58{\times}10^{-3}\;day^{-l}$ for OMP-ED suppository, respectively. Additives such as arginine, eglumine and ED had some stabilizing effect for OMP-HPCD, OMP-CHL and OMP-Na suppositories, respectively. After 6 month-storage at $30^{\circ}C$, 75% RH, OMP-CHL suppository was most stable. The values of Tmax for OMP-HPCD and OMP-Na suppositories were $11.7{\pm}2.36\;and\;11.4{\pm}2.56\;min$, respectively. The values of Cmax for OMP-HPCD and OMP-CHL suppository were $2.31\;{\mu}g/ml\;(p<0.01)\;and\;1.89\;{\mu}g/ml\;p<0.01)$, respectively. The values of AUC for OMP and $OMP-{\beta}-CD$ suppository were $61.9{\pm}25.79\;and\;68.6{\pm}29.48\;{\mu}g\;{\cdot}\;min/ml$, and the corresponding values for OMP-HPCD and OMP-CHL were $106.1{\pm}43.16\;(p<0.05)\;and\;127.3{\pm}42.52\;{\mu}g\;{\cdot}\;min/ml(p<0.01)$, respectively. The above results indicate the OMP-HPCD and OMP-CHL suppositories have the excellent bioavailabilties in vivo study.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.

• 생명과학회지
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• 제18권2호
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• pp.241-248
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• 2008

축산물을 이용하여 생산된 특이항체는 세균성감염에 의한 설사병 치료와 충치예방에 효과가 있고, 특히 계란을 이용한 IgY (Immunoglobulin Yolk)는 비교적 산과 열에 안정하다는 실험결과가 보고되어[22] 있다. 식품분야에 특이항체를 식품소재로 산업화에 활용한 빈도는 아직 미미한 상태에 있으나, 최근 면역학, 단백질공학, 생명공학 등의 발전과 기술 향상으로 식품 소제로써의 활용성이 점차 높아질 것으로 기대된다. 본 연구에서는 H. pylori의 감염을 예방하고 치료보조제로 사용할 목적으로 포유동물을 통한 피동면역용 항체를 생산하고자 하였다. H. pylori로부터 항원을 분리하고, 분리된 항원에 대한 항체생산 및 H. pylori의 응집정도를 알아보았다. H. pylori로부터 분리된 주요 항원 단백질은 WC, OMP, crude urease, LPS 각각 12개, 7개, 3개, 1개 종류의 band를 확인할 수 있었다. 분리된 항원의 IgG 항체 생성은 동일한 항원농도인 $20{\mu}g/100{\mu}l$에서 각각 WC (L) $77.9{\pm}6.4{\mu}g/ml$, OMP $84.9{\pm}6.4{\mu}g/ml$, crude urease $123.8{\pm}2.9{\mu}g/ml$로 crude urease 항원이 가장 많은 것으로 나타났다. 그리고 IgA 항체 생성은 WC (L) $2.5{\pm}0.32{\mu}g/ml$, OMP $2.0{\pm}0.43{\mu}g/ml$, crude urease $1.3{\pm}0.25{\mu}g/ml$로 IgA 항체 생성은 WC (L) 항원이 가장 많은 것으로 나타났다 Western blotting을 통하여 항원 단백질의 면역원성 알아본 결과 WC 10종류, OMP 6종류, crude urease 3종류의 주요 항원성 물질을 확인할 수 있었다. 항체의 H. pylori에 대한 응집정도를 나타내는 응집가는 anti-WC (H), anti-WC (L), anti-OMP, anti-crude urease 항혈청에서 각각 $2^5,\;2^5,\;2^6\;and\;2^7$으로 나타났으며, 상대적으로 anti-crude urease 항혈청이 가장 높은 응집가를 나타내었다. 각 항원에 의해 생성된 항혈청의 urease활성 억제에 대한 흡광도(OD=550 nm)는 WC (H) $0.14{\pm}0.01$, WC (L) $0.16{\pm}0.01$, OMP $0.18{\pm}0.03$, Urease $0.18{\pm}0.04$로 대조구 $0.26{\pm}0.02$와 비교할 때 유의적인 억제효과가 있는 것으로 나타났다. 이상의 결과를 종합해 볼 때 H. pylori 항원의 분리와 분리된 항원의 항체 생성능, H. pylori의 응집가, urease 활성억제측면에서 WC 및 crude urease항원 모두 높은 항체 역가를 나타내었다.

• 한국어병학회지
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• 제12권2호
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• pp.89-99
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• 1999

V. vulnificus ATCC 27562균주의 배양 온도를 $2^{\circ}C $, 20분간 및 6% 에탄올, 10분간으로 반응시켰을 때 SDS-PAGE상에서 새로운 16가지의 heat shock protein(hsps)과 10가지의 ethanol shock protein이 나타났다. Lethal temperature에 노출하기전에 미리 열 충격을 가한 경우 thermo tolerance가 유도되었다. 균체면역에 의해 생성된 항혈청과 열 충격 세포에서 분리된 막단백질과의 ELISA에서는 Outer Membrane Protein(OMP)에서 높은 면역반응을 나타냈으며 western blotting으로는 Inner Membrane Protein(IMP)에서는 62kDa, OMP에서는 69 kDa단백이 높은 면역원성을 나타냈다. ethanol 충격 반응에서는 IMP에서는 48 kDa, OMP에서는 오직 major밴드에서만 면역반응성이 확인되었다. anti-V, vulnificus혈청에 대한 균체 응집시험에서는 열 충격 반응 후의 균체가 정상 균체에 비해 응집반응성이 높았다.

• 생명과학회지
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• 제14권6호
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• pp.986-990
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• 2004

2002년 5월에서 7월사이 한국 근해로부터 총 가검물 50례 중에서 V. vulnificus은 5균주가 분리되었고, API 20E kit의동정률이 $89.4\~93.7\%$로 나타났다. V. vulnificus 16S rDNA를 증폭하여 얻은 산물을 cloning하여 염기서열을 결정하고 분석한 결과 V. vulnificus로 확인되었다. 동정된 분리균주들의 cell lysates을 SDS-PAGE로 분석하였고 표준균주로 사용된 V. vulnificus ATCC 27562와 분리 균주들을 비교해 본 결과 단백밴드pattern이 많은 차이를 보였으나 외막단백질은 V. vulnificus간에는 공통의 밴드가 확인되었으나 V. parahaemolyticus 와는 차이를 보였다.

• 한국동물위생학회지
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• 제32권4호
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• pp.335-341
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• 2009

Canine brucellosis produce abortions and infertility in dogs and is currently diagnosed by serological methods such as rapid slide agglutination test with 2-mercaptoethanol (2-ME RSAT) and immunochromatographic assay (ICA). Bacterial isolation is considered gold standard for Brucella diagnosis and the polymerase chain reaction (PCR) is an alternative method to bacterial isolation. A total of 36 whole blood samples were collected from dogs reared in area of Chuncheon and were subjected to serology (2-ME RSAT and ICA for B. canis, Rose Bengal test and C-ELISA for B. abortus), blood culture and 3 types of PCRs (BSCP31, 16s rRNA, and OMP-2). All blood samples were negative by serology and blood cultures. The BCSP31 and the OMP-2 PCR detected 5 samples were positive whereas the 16S rRNA PCR detected all samples were negative as serological methods and blood culture did. From the results observed in the present study, we conclude that 16S rRNA PCR could be used for direct PCR for canine blood samples.

• 대한수의학회지
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• 제32권2호
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• pp.181-194
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• 1992

Two hundred and eighty nine strains of Yersinia enterocolitica isolated from healthy pigs were tested for the presence of 40~50 Megadalton virulence-associated plasmids and plasmidmediated in vitro virulence-associated properties, i.e., congo red uptake, calcium dependency, autoagglutination, CRMOX reaction, crystal violet binding and pyrazinamidase reaction. The correlationships between in vitro virulence-associated properties and the presence of 220 Kdalton outer membrane protein(OMP) were examined in strains with or without virulence-associated plasmids. The correlationships between the presence of plasmids on the production of the OMP and the expression of in vitro virulence-associated properties were studied with $CRMOX^+$ strains and acridine orangecured $CRMOX^-$ mutants. The results were as follows : 1. Of the in vitro virulence-associated tests with 289 strains of Y enterocolitica, 275 strains (95.2%) were positive for pyrazinamidase test, and followed by in order of crystal violet binding test, 226 (79.2% ) ; CRMOX test, 190 (65.7%) ; autoagglutination test, 1.85(64.0%) : calcium dependency test, 86 (29.8%) and congo red uptake test, 47(16.3%). 2. The correlationship between autoagglutination and CRMOX test(r=0.90) was highly significant (p<0.01). 3. In 190 strains(65.7%) bearing the virulence-associated plasmids(MW 40~50 Mdalton), the correlation between the presence of plasmids and their in vitro virulence-associated properties were highest with CRMOX test(r=0.93) and followed by in orders of AAG test(0.81), CV test(0.46), PYZ test(0.37) and CD test(0.18), but no correlationship between the presence of plasmids and CR test(-0.11). 4. The $CRMOX^+$ strains produced the 220 Kdalton OMP when they were cultured at $37^{\circ}C$, but not at $26^{\circ}C$. The presence of 220 Kdalton OMP was correlated significantly with in vitro virulence properties and the presence of virulence-associated plasmid, respectively. 5. In the isogenic $CRMOX^-$ mutant strains, of which plasmid were cured by treatment with acridine orange not only in vitro virulence-associated properties(CR 100%, CD 100%, AAG 82.6%, CV 58.3%) disappeared but also 220 Kdalton OMP(100%) was not produced. These results indicate that the positive CRMOX reaction is plasmid-mediated and the CRMOX test is potential as an in vitro virulence tests with Y enterocolitica.

• ETRI Journal
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• 제42권3호
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• pp.376-387
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• 2020

Massive computation of the reconstruction algorithm for compressive sensing (CS) has been a major concern for its real-time application. In this paper, we propose a novel high-speed architecture for the orthogonal matching pursuit (OMP) algorithm, which is the most frequently used to reconstruct compressively sensed signals. The proposed design offers a very high throughput and includes an innovative pipeline architecture and scheduling algorithm. Least-squares problem solving, which requires a huge amount of computations in the OMP, is implemented by using systolic arrays with four new processing elements. In addition, a distributed-arithmetic-based circuit for matrix multiplication is proposed to counterbalance the area overhead caused by the multi-stage pipelining. The results of logic synthesis show that the proposed design reconstructs signals nearly 19 times faster while occupying an only 1.06 times larger area than the existing designs for N = 256, M = 64, and m = 16, where N is the number of the original samples, M is the length of the measurement vector, and m is the sparsity level of the signal.

• 한국수산과학회지
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• 제51권3호
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• pp.254-261
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• 2018

From 2014 to 2017, mortalities of seawater-cultured rainbow trout Oncorhynchus mykiss, were observed in the Goheung and Jeju areas of Korea, with Vibrio anguillarum (seven strains: RT1, 2, 3, 4, 5, 6, and 7) identified as the etiological agent. The phenotypic (based on API 20NE, API ZYM, and E-test kits), serotypic (slide agglutination tests with O1, O2, O3, O4, and O7 antisera), and genotypic (16S rRNA and ompU sequencing) characteristics of the seven RT strains were analyzed and compared to those of seven additional V. anguillarum stains (SF, isolated from sweet fish; FM, isolated from flathead mullet; ATCC43305; ATCC43311; ATCC43307; ATCC43308; and KCTC2711). The phenotypes of the RT strains showed variance, while the slide agglutination tests of the RT1-7, SF, and FM strains all showed positive reactions with serotype O1 antiserum. The 16S rRNA and ompU sequences of the RT1-7, SF, and FM strains were affiliated with V. anguillarum ATCC43305 (Serotype O1), but the ompU sequence of the SF strain differed from those of the RT1-7, FM, and ATCC43311 strains, including one amino acid substitution. We thus confirmed that serotype O1 V. anguillarum, with multiple phenotypes, continues to infect seawater-cultured rainbow trout in Korea.