• Clinical and Experimental Pediatrics
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• v.50 no.2
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• pp.143-150
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• 2007

Purpose : This study was conducted to validate enzyme immunoassay (EIA) for the quantitative measurement of human IgG antibodies specific for Haemophilus influenzae type b (Hib) capsular polysaccharide. Method : We evaluated specificity, repeatability, intermediate precision, accuracy, lower limit of quantification (LLOQ), and stability to validate standardized EIA for the quantitative measurement of human anti-polyribosylribitol phosphate (PRP) IgG antibodies. Results : The results indicated that this EIA showed specificity to HbO-HA antigen and repeatability and intermediate precision were within acceptance criteria (repeatability: $CV{\leq}15%$, intermediate precision: $CV{\leq}20%$). The EIA-derived results from this laboratory were equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-PRP antibodies in the 28 sera. Spiking recovery result was within acceptance criteria ($100{\pm}20%$). The precision and accuracy of samples in LLOQ were from -14.7 to -4.7% in nominal values, which were within acceptance criteria (precision: $CV{\leq}25%$, accuracy: ${\pm}25%$). Freeze-thaw stability and short term temperature stability were within ${\pm}20%$ of acceptance criteria. Conclusions : The EIA which is performed at the Center for Vaccine Evaluation and Study Ewha Medical Research Institute, is an appropriate serologic assay which can be used for quantitation of anti-PRP IgG antibodies in human sera.

• Biomolecules & Therapeutics
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• v.18 no.3
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• pp.271-279
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• 2010

Streptococcus pneumoniae is the major pathogen that frequently causes serious infections in children, the elderly and immunocompromised patients. S. pneumoniae is known to produce reactive oxygen species (ROS) and S. pneumoniae-produced ROS is considered to play a role in pneumococci pathogenesis. SIGN-R1 is the principal receptor of capsular polysaccharides (CPSs) of S. pneumoniae. However, there is a considerable lack of knowledge about the protective role of SIGN-R1 against S. pneumoniae-produced ROS in SIGN-$R1^+$ macrophages. While investigating the protective role of SIGN-R1 against ROS, we found that SIGN-R1 intimately bound to peroxiredoxin-1 (Prx-1), one of small antioxidant proteins in vitro and in vivo. This interaction was increased with ROS generation which was produced by stimulating SIGN-R1 with dextran, a polysaccharide ligand of SIGN-R1. Also, SIGN-R1 crosslinking with 22D1 anti-SIGN-R1 antibody increased Prx-1 in vitro or in vivo. These results suggested that SIGN-R1 stimulation with CPSs of S. pneumoniae increase the expression level of Prx-1 through ROS and its subsequent interaction to SIGN-R1, providing an important antioxidant role for the host protection against S. pneumoniae.

• YAKHAK HOEJI
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• v.48 no.6
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• pp.345-351
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• 2004

ln the present work to determine effect of a Streptococcus pneumoniae conjugate vaccine, S.pneumoniae capsule attached to the surface protein (JY-Pol) was ex amined. This JY-Pol contained approximately 92% and 6% carbohydrate and protein, respectively. Gel electrophoresis revealed the presence of the surface protein in the JY-Pol. By the double immunodiffusion and isotyping ELISA analyses, administration of JY-Pol that was adsorbed to alum adjuvant (JY-Pol/Alum) into mice induced IgM, IgG, and IgA specific for the S.pneumoniae capsule. The ATCC capsular polysaccharide adsorbed to alum (ATCC-Pol/Alum) provoked only IgM in mice. In survival tests, mice that were immunized with the JY-Pol/Alum before intravenous challenge with live S.pneumoniae survived entire period of 46 day-observation, whereas all mice that received ATCC-Pol/Alum or only diluent instead of the vaccination died within 5 and 12 days, respectively. Results from footpad-edema test showed that JY-Pol/Alum formula provoked the cellular immunity as determined by swelling of the mouse footpad. These data indicate that the naturally conjugated JY- Pol enhances resistance of mice against disseminated pneumococcal disease due to S.pneumoniae by both humoral and cellular immune responses.

• Molecules and Cells
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• v.21 no.1
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• pp.82-88
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• 2006

The global pattern of growth-dependent gene expression in Streptococcus pneumoniae strains was evaluated using a high-density DNA microarray. Total RNAs obtained from an avirulent S. pneumoniae strain R6 and a virulent strain AMC96-6 were used to compare the expression patterns at seven time points (2.5, 3.5, 4.5, 5.5, 6.0, 6.5, and 8.0 h). The expression profile of strain R6 changed between log and stationary growth (the Log-Stat switch). There were clear differences between the growth-dependent gene expression profiles of the virulent and avirulent pneumococcal strains in 367 of 1,112 genes. Transcripts of genes associated with bacterial competence and capsular polysaccharide formation, as well as clpP and cbpA, were higher in the virulent strain. Our data suggest that late log or early stationary phase may be the most virulent phase of S. pneumoniae.

• Journal of Animal Science and Technology
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• v.63 no.3
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• pp.662-665
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• 2021

Streptococcus suis is a major pig pathogen causing severe economic losses to the swine industry. This study aimed to analyze the genome of S. suis strain INT-01 isolated from a domestic pig in Korea. We found that the genome of strain INT-01 contains 2,092,054 bp, with a guanine (G) + cytosine (C) content of 41.3%, and the capsular polysaccharide synthesis locus of this strain is almost identical to that of serotype 3 S. suis strain 4961 isolated from China, suggesting that these isolates can be classified as serotype 3. Genomic analyses revealed that strain INT-01 is an extracellular protein factor (epf)^-/ muraminidase-released protein (mrp)⁺/ suilysin (sly)^- S. suis, which is the most prevalent genotype in Korea, and several virulence-related genes associated with the pathogenicity of S. suis were also detected. The genomic information of strain INT-01 may provide important insights into the development of control strategies against S. suis infections in Korea.

• Clinical and Experimental Pediatrics
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• v.56 no.7
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• pp.286-290
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• 2013

Purpose: Although chronic and recurrent rhinosinusitis is prevalent in children, little is known about its causes. Here, we investigated the humoral immunity in children with chronic or recurrent rhinosinusitis. Methods: We examined 16 children attending the outpatient clinic at the CHA Bundang Medical Center including 11 boys and 5 girls, aged 3-11 years (mean age, 5.6 years), who had rhinosinusitis for >3 months or >3 times per year. The complete blood count with differential and total serum concentrations of Immunoglobulin (Ig) E, IgA, IgD, IgM, IgG, and IgG subclasses ($IgG_1$, $IgG_2$, $IgG_3$, and $IgG_4$) of all children were measured. All subjects received 23-polysaccharide pneumococcal vaccination (PPV), and the levels of antibodies to 5 serologic types (4, 6B, 14, 18C, and 23F) of pneumococcal capsular polysaccharide antigens were measured before and after vaccination. Post-PPV antibody titers ${\geq}0.35{\mu}g/mL$ or with a ${\geq}4$-fold increase were considered as positive responses. Results: The titers of IgG, IgA, IgD, and IgM were within normal range in all 16 children, whereas the total IgE concentration was higher than normal in 2 children. $IgG_1$ deficiency was observed in 1 patient and $IgG_3$ deficiency in 3. After PPV, 1 patient failed to respond to all 5 serologic types, 2 failed to respond to 4 serologic types, and 2 failed to respond to 3 serologic types. Conclusion: Clinicians should consider the evaluation of humoral immune functions in children with chronic or recurrent rhinosinusitis who do not respond to prolonged antibiotic treatment.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.247-253
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• 2003

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.63-71
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• 2000

An immunogenic, high molecular weight lipopolysaccharide (LPS)-protein complex isolated from a potassium thioncyanate extract of a Pasteurella multocida (P multocida ; strain P-2383, capsular type A and somatic type 3) was characterized. Chemical analysis of the complex by gas chromatography on a capillary column demonstrated that this complex contained most of the chemical constituents characteristic of LPS extracted by the phenol-water methed from the whole bacterium. However, there was proportionately more carbohydrate than fatty acid in the complex in contrast to LPS in which fatty acid seemed to be in excess. When toxicity of the complex was evaluated in 10-day-old chicken embryos, the complex was less toxic ($LD_{50}=12.72{\mu}g$) than the purified LPS ($LD_{50}=0.44{\mu}g$). The $LD_{50}$, of the LPS moiety extracted from the complex was $5.24{\mu}g$. Composition of the complex was analyzed by SDS-PAGE with silver staining and Western immunoblotting. The complex did not migrate through the polyacrylamide gel unless dissociated with SDS. The complex dissociated with SDS contained at least 32 different protein and polysaccharide components: 18 components reacted with an antiserum against the complex. There was no significant compositional variation between the complexes from different strains, but quantitative differences in individual components were noted. When cross-protectivity of the complex was evaluated in mice, this complex provided substantial protection not only against the homologous bacteriun but also against different P multocida strains of the same serotype. LPS-protein complexes isolated by the same method from other strains also induced protection against an challenge with P-2383.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.665-680
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• 1996

Bovine Pneumonic Pasteurellosis는 수송열(輸送熱)로 일반적으로 알려져 있는 질병으로서, 여러가지 요인의 복합적(複合的)인 작용에 의해 발병하는 것으로 알려져 있으나, Pasteurella haemolytica A1이 가장 주요(主要)한 인자(因子)로 밝혀져 있다. P haemolytica A1은 leukotoxin(LKT), lipopolysaccharide(LPS), capsular polysaccharide 등 여러가지의 병원성인자(病原性因子)을 생성한다. 이들 인자중 LKT가 가장 중요한 병원성인자로 밝혀져 있다. 이에 본 실험은 P haemolytical A1의 LKT 유전자를 Bacillus subtilis에서 발현(發現)시킴으로서 LPS에 오염(汚染)되지 않은 LKT을 대량으로 생산할 목적으로 실시되었다. 실험의 첫 단계(段階)로서 pLKT52 plasmid을 Sau3 A1의 제한효소을 이용하여 부분소화(部分消化)시킨 후 이 부분 소화(消化)된 유전자들로부터 3~5kb 크기의 유전자들을 순수분리하여 pUC18와 결합시킨 후 E coli NM522에 형질전환(形質轉換)시켰다. 이때 형질전환된 균주들은 LKT에 대한 단크론 항체인 MAb601을 이용하여 colony blot 법에 의해서 LKT 유전자 보유 및 발현여부(發現與否)을 조사하였다. 이들 양성 clone들은 제한효소분석(制限酵素分析), 염기서열분석(鹽基序列分析) 및 Western blot 등에 의해서 재확인(再確認)하였다. 총 9개의 양성 clone중 위의 방법에 의해서 한 clone을 선택(選擇)하여 lktCA insert를 재분리하여 shuttle vector에 subcloning 하였다. Subcloning된 LKT 유전자들은 shuttle vector의 종류(種類)(pHPS9, p602/20, pHPS9-Sac)와 각기(各其) 다른 종류(種類)의 B subtilis(spoO12A, BR121, WB3O, Raj1105) 숙주내(宿主內)에서 발현정도를 Western blot 법에 의해서 비교(比較)하였다. 이때 최적발현조건(最適發現條件)은 p602/20와 pBL1의 dual plasmid system을 이용하여 B subtilis spoO12A에서 2시간동안 IPTG로 발현을 유도(誘導)하는 것이었다. B subtilis에서 발현된 LKT을 visual 법과 neutral red uptake 법을 이용하여 소 폐포(肺胞) 대식구(大食求)에 대한 biological activity를 확인하였다. 발현된 LKT에 대한 LPS 오염은 LKT을 SDS-PAGE 후 silver stain에 의해서 확인하였다. 본 실험을 통해서 볼 때에 lktCA 유전자를 보유(保有)하고 있는 p602/20는 B subtilis에서 매우 불안정(不安定)하였고, 발현된 LKT는 세균자체(細菌自體)에서 생성되는 protease들에 의해서 파괴(破壞)됨으로서 농도(濃度)가 매우 낮았다. 이러한 문제점들은 다음 단계(段階)의 실험에서 해결되어야할 문제들이다.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.448-452
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• 2018

The full genome sequence of Bacillus safensis KCTC 12796BP which had been isolated from the marine sponge in the seawater of Jeju Island, was determined by Pac-Bio next-generation sequencing system. A circular chromosome in the length of 3,935,874 bp was obtained in addition to a circular form of plasmid having 36,690 bp. The G + C content of chromosome was 41.4%, and that of plasmid was 37.3%. The number of deduced CDSs in the chromosome was 3,980, whereas 36 CDS regions were determined in a plasmid. Among the deduced CDSs in chromosome, 81 tRNA genes and 24 rRNA genes in addition to one tmRNA were allocated. More than 30 CDSs for sporulation, 16 CDSs for spore coat, and 20 CDSs for germination were also assigned in the chromosome. Several genes for capsular polysaccharide biosynthesis and for flagella biosynthesis and chemotaxis in addition to genes for osmotic tolerance through glycine-choline betaine pathway were also identified. Above all, the biosynthetic gene cluster for anti-allergic compounds seongsanamides were found among two non-ribosomal peptide synthetase (NRPS) gene clusters for secondary metabolites.