• Asian Journal of Atmospheric Environment
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• 제6권1호
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• pp.33-40
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• 2012

In this study, we characterized the physical form of allergenic Cry j 1 in the urban atmosphere. Through an immunofluorescence antibody method, we showed that allergenic Cry j 1 exists as fine particles (${\leq}1.1{\mu}m$). To determine Cry j 1 concentrations and its particle size distribution, we used the ELISA method to confirm that most Cry j 1 exists as fine particles in the urban atmosphere and is found at high concentrations on fine day next to rainy day. Furthermore, we evaluated Cry j 1 denaturation by using the Biacore J system based on the surface plasmon resonence (SPR) principle and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We showed that the dissociation constant ($K_D$) of Cry j 1 that has been exposed to urban polluted air is lower ($1.76{\times}10^{-14}$ M) than that of Cry j 1 ($1.32{\times}10^{-9}-3.37{\times}10^{-9}$ M) of original pollen grains that has not been exposed to air pollutants. Cry j 1 turns into low molecular weight proteins by reacting with various acidic solutions. In sum, we showed that allergenic Cry j 1 exists as fine particles that can deposit in the lower respiratory tract. This finding clarifies the relationship between Japanese cedar pollinosis and air pollutants.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.809-815
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• 2018

Influenza, which is a highly contagious disease caused by the influenza A virus, continues to be a major health concern worldwide. Although the accurate and early diagnosis of influenza virus infection is important for controlling the spread of this disease and rapidly initiating antiviral therapy, the current influenza diagnostic kits are limited by their low sensitivity. In this study, we developed several new influenza nucleoprotein (NP)-specific monoclonal antibodies (mAbs) and compared their sensitivity and specificity of those with commercially available anti-NP mAbs. Three mAbs, designated M24.11, M34.3, and M34.33, exhibited higher reactivities to recombinant NPs and A/Puerto Rico/8/1934 (H1N1) viral lysates compared with the commercial mAbs, as assessed using enzyme-linked immunosorbent assays. M34.3 and M34.33 showed higher reactivities with A/California/04/09 (pandemic H1N1) and A/Philippines/2/82 (H3N2) viral lysates than the commercial mAbs. In contrast, M24.11 had marked reactivity with H3N2 but not with pandemic H1N1. Immunofluorescent confocal microscopy showed that the three mAbs effectively detected the presence of influenza virus in lung tissues of mice infected with A/Puerto Rico/8/1934. These results indicate that the newly developed M34.3 and M34.33 mAbs could be useful for the development of influenza diagnostics.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.290-295
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• 1994

30 monoclonal antibodies (mAbs) were produced against Bacillus thuringiensis subsp. canadensis. Out of the these, 6 mAbs were selected for further studies. SDS-PAGE analyses of sonicated antigens of 10 8. thuringiensis strains showed that they generally had both predominant protein antigens of molecular weights of 45 kilodalton (kd) except for shandogiensis and konkukian, and 37kd except for israelensis, tochigiensis, and shandogiensis, respectively. These results indicate that 4kd and 37kd may be important for demonstrating common antigens except for a few strains of B. thuringiensis. In comparing the result of the westem blot using mAbs with that of using polyclonal antibodies to canadensis, we found that immunoreactive proteins of 99 and 39 kd were identified as common antigens, which might act as antigenic determinants, and might be surface or flagella antigens. Reactivities of mAbs with 41 strains of 8. thuringiensis demonstrated that mAbs of C-1, C-3, C-4, C-S and C-6 except C-2 did not recognize epitopes of thuringiensis, but that all of the mAbs recognized epitopes of galleriae, kurstaki, dakota, tolrJokuensis, silo, toguchini, and leesis. The potential applications of the mAbs we produced would be useful tools for the clarification of taxonomy, investigation of antigenic relationship between B. thuringiensis strains, and localization of specific surface and flagella antigens.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1621-1628
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• 2015

Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.248-254
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• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.352-356
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• 2007

A Lactobacillus sp. has been isolated from infant feces and characterized according to its physiological properties and identified as Lactobacillus acidophilus KLA1012. A gene coding surface layer protein (SLP) has been cloned and the sequence has been determined. The nucleotide sequence of slpA was 1,338 bp in size and was identical to that of L. acidophilus ATCC 4356 (100%). Amino acid sequence of SLP-A was deduced from the nucleotide sequence and it had signal sequence at N-terminal, consisting of positively charged amino acid mainly lysine. slpA was cloned and heterologously expressed in E. coli M15 and the 45.2 kDa surface-layer protein band was examined by SDS-PAGE and confirmed by Western blotting using polyclonal antibody against L. acidophilus KLA 1012 SLP-A protein.

• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.965-974
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• 2017

The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, $55.2{\pm}0.3^{\circ}C$, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, $52.5{\pm}0.3^{\circ}C$, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

• 대한수의학회지
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• 제38권2호
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• BMB Reports
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• 제38권5호
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• pp.595-601
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• 2005

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7% and 61.9% identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2% identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both $Ca^{2+}$ and calmodulin. The GAD-encoded 56~58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a $Ca^{2+}$/calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

• Journal of Plant Biotechnology
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• 제36권1호
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• pp.68-74
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• 2009

B. rapa로부터 분리한 BrMT3 유전자를 도입시킨 효모와 애기장대가 카드뮴을 비롯한 중금속에 저항성을 보이는 것이 확인되었고 이 결과를 토대로 이 유전자가 중금속 흡착을 통한 환경 정화 및 스트레스에 내성을 갖는 형질전환 식물체를 개발하는데 유용하게 사용될 것으로 기대된다.