• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1047-1054
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• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• BMB Reports
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• 제31권1호
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• Journal of Periodontal and Implant Science
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• 제29권4호
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• pp.953-964
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• 1999

본 연구는 세 종류의 Porphyromonas gingivalis(Pg) antigen의 IgG subclass associated recognition을 평가하기 위해 수행했었다. 총 35명의 조기발병형치주질환자중, Pg381에 대한 IgG2항체의 증가를 보이는 5명이 급속진 행형 치주질환자, IgG4의 증가를 보이는 6명의 환자(국소유년형 치주질환자 2명과 급속진행 형 치주질환자 4명), IgG2+4의 증가를 보이는 2명의 급속진행형 치주질환자 그리고 IgG1+2+4의 증가를 보이는 8명의 환자(국소 유년형 치주질환자 2명과 급속진행형 치주질환자 6명)으로 구성된 21명의 환자를 dot immunoblot analysis를 위해 선택했다. 실험에 사용된 정제된 항원은 Pg381에서 추출한 43-kd fimbrilin protein과 lipoplysaccharide(LPS), Pg A7A1-28(ATCC 53977)에서 추출한 capsularpolysaccharide(CPS)였다. Immunoblotting pattern은 IgG4 antibody가 fimbrial antigen에 강력히 반응함을 보여주었다. Fimbriae에 잘 반응하는 몇몇의 IgG4 antibody역시 antigen에 대해 양성반응을 보였다. 대조적으로 IgG2는 CPS antigen을 일차적으로 인식했다. 전부는 아니지만 대부분의 경우, single이나 group화된 IgG subclass는 모두 LPS antigen을 인식하지 못했다. 같은 group에서 염색강도의 개인적인 차이는 증명되었다. 이런 결과는 조기발 병형 치주질환에서 Pg의 fimbriae와 CPS가 immunodominant antigen이 될 수 있음을 제시한다. 더욱이 IgG subclass antibody가 이런 Pg의 immunoglobulin antigen을 선택적으로 인식함을 알았고, 이는 조기발병형치주질환의 병리에 immunodominant antigen과 함께 IgG의 기능적인 역할을 고려해야 함을 제시한다.

• IMMUNE NETWORK
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• 제24권3호
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• pp.19.1-19.15
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• 2024

The influenza virus poses a global health burden. Currently, an annual vaccine is used to reduce influenza virus-associated morbidity and mortality. Most influenza vaccines have been developed to elicit neutralizing Abs against influenza virus. These Abs primarily target immunodominant epitopes derived from hemagglutinin (HA) or neuraminidase (NA) of the influenza virus incorporated in vaccines. However, HA and NA are highly variable proteins that are prone to antigenic changes, which can reduce vaccine efficacy. Therefore, it is essential to develop universal vaccines that target immunodominant epitopes derived from conserved regions of the influenza virus, enabling cross-protection among different virus variants. The internal proteins of the influenza virus serve as ideal targets for universal vaccines. These internal proteins are presented by MHC class I molecules on Ag-presenting cells, such as dendritic cells, and recognized by CD8 T cells, which elicit CD8 T cell responses, reducing the likelihood of disease and influenza viral spread by inducing virus-infected cell apoptosis. In this review, we highlight the importance of CD8 T cell-mediated immunity against influenza viruses and that of viral epitopes for developing CD8 T cell-based influenza vaccines.

• IMMUNE NETWORK
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• 제15권4호
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• pp.186-190
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• 2015

Cytomegalovirus (CMV) infection in healthy individuals is usually asymptomatic and results in latent infection. CMV reactivation occasionally occurs in healthy individuals according to their immune status over time. T cell responses to CMV are restricted to a limited number of immunodominant epitopes, as compared to responses to other chronic or persistent viruses. This response results in progressive, prolonged expansion of CMV-specific $CD8^+$ T cells, termed 'memory inflation'. The expanded CMV-specific $CD8^+$ T cell population is extraordinarily large and is more prominent in the elderly. CMV-specific $CD8^+$ T cells possess rather similar phenotypic and functional features to those of replicative senescent T cells. In this review, we discuss the general features of CMV-specific inflationary memory T cells and the factors involved in memory inflation.

• Journal of Microbiology and Biotechnology
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• 제5권6호
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• pp.353-358
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• 1995

Escherichia coli causes intestinal and extraintestinal infections and has been an indicator of fecal pollution in water and food. BALB/c mouse was immunized by injection of somatic E. coli (ATCC 8739) cells to produce monoclonal antibodies. Splenocytes of mouse were fused with myeloma cells (Sp2/0-Ag14). Two hybridomas secreting monoclonal antibodies were established after being cloned. In SDS-PAGE analysis of E. coli antigens 37 protein profiles appeared from 14 kDa to 182 kDa. Western blot analysis using polyclonal antibodies demonstrated that protein antigens of 41 kDa, 38.2 kDa and 31.7 kDa were immunodominant. Monoclonal antibodies DY-CM1 and DY-CM2 recognized 31.7 kDa and 2.0 kDa antigens in Western blot analysis, respectely.

• BMB Reports
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• 제49권6호
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• pp.331-336
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• 2016

In murine collagen-induced arthritis (CIA), self-reactive T cells can recognize peptide antigens derived from type-II collagen (CII). Activation of T cells is an important mediator of autoimmune diseases. Thus, T cells have become a focal point of study to treat autoimmune diseases. In this study, we evaluated the efficacy of recombinant MHC class II molecules in the regulation of antigen-specific T cells by using a self peptide derived from CII (CII260-274; IAGFKGEQGPKGEPG) linked to mouseI-A^q in a murine CIA model. We found that recombinant I-A^q/CII260-274 molecules could be recognized by CII-specific T cells and inhibit the same T cells in vitro. Furthermore, the development of CIA in mice was successfully prevented by in vivo injection of recombinant I-A^q/CII260-274 molecules. Thus, treatment with recombinant soluble MHC class II molecules in complex with an immunodominant self-peptide might offer a potential therapeutic for chronic inflammation in autoimmune disease such as rheumatoid arthritis.

• The Plant Pathology Journal
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• 제27권4호
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• pp.367-371
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• 2011

Multiplex PCR assays were developed for the simultaneous detection of ten important Korean quarantine phytoplasmas. The species-specific primers were designed based on ribosomal protein, putative preprotein translocase Y, immunodominant protein, elongation factor TU, chaperonin protein and the 16S rRNA genes of 'Candidatus (Ca.) Phytoplasma' species. Three main primer sets were prepared from ten designed primer pairs to limit nonspecific amplification as much as possible. The multiplex PCR assay using the three primer sets successfully amplified the correct conserved genes for each 'Ca. Phytoplasma' species. In addition, ten important 'Ca. Phytoplasma' species could be easily determined by recognizing band patterns specific for each phytoplasma species from three primer sets. Moreover, a high sensitivity of multiplex PCR for each primer set was observed for samples containing a low DNA concentration (10 ng/${\mu}l$). This study provides the useful multiplex PCR assay as a convenient method to detect the presence of ten important quarantine phytoplasmas in Korea.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.173-178
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• 2001

A monoclonal antibody (mAb) to the glucoprotein D (gD) of Herpes simplex virus type 2 (HSV-2) was successfully generated by hybridoma technology and characterized. The mAb, SKS2v, recognized a gD antigen with an apparent molecular mass of 60kDa in a Western blot analysis. The isotype was determined by a sandwich ELISA to be IgG2a. HSV-2 exhibited major antigens of 36, 43, 46, 47, 60, 69, 81, 96, 109, 112, 159, and 227 kDa among 25 protein profiles in SDS-PAGE, and among these antigens, those of 60, 112, 125, and 227 kDa were immunodominant in a Western blot analysis using antisera, thereby indicating that they play a role in inducing neutralizing antibodies in HSV-2 infection. When reacted with Vero cells infected with HSV-1 and HSV-2 SKSv2 showed a reactivity to the surface of the infected cells, and a gD antigen of 60 kDa appeared to be expressed in both types of HSV.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.431-437
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• 2016

The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.