• BMB Reports
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• 제46권7호
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• pp.358-363
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• 2013

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein.

• 생명과학회지
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• 제9권3호
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• Journal of Microbiology
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• 제42권2호
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• pp.126-132
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• 2004

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30$^{\circ}C$ until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30$^{\circ}C$ for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Topl0F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, com-pared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

• Reproductive and Developmental Biology
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• 제42권2호
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• pp.7-12
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• 2018

In this review, we have tried to summarize the evidence and molecular characterization indicating that $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) is a group of the aldo-keto reductase (AKR) family, and it plays roles in the modulation and regulation of steroid hormones. This enzyme plays a critical role in the regulation of luteal function in female mammals. We have studied the molecular expression and regulation of $20{\alpha}$-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine $20{\alpha}$-HSD was generated in a rabbit immunized with the purified recombinant protein. The mRNA expression levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. The mRNA was also specifically detected in the placental and ovarian tissues during pregnancy. The $20{\alpha}$-HSD protein was intensively localized in the large luteal cells and placental cytotrophoblast villus, glandular epithelial cells of the endometrium, syncytiotrophoblast of the placenta, the isthmus cells of the oviduct, and the basal part of the primary chorionic villi and chorionic stem villus of the placenta and large luteal cells of the CL in many mammalian species. Further studies are needed to determine the functional significance of the $20{\alpha}$-HSD molecule during ovulation, pregnancy, and parturition. This article will review how fundamental information of these enzymes can be exploited for a better understanding of the reproductive organs during ovulation and pregnancy.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.489-499
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• 2019

Subunit vaccines are safer and more stable than live vaccines although they have the disadvantage of eliciting poor immune response. To develop a subunit vaccine, an effective delivery system targeting the key elements of the protective immune response is a prerequisite. In this study, oxidized carbon nanospheres (OCNs) were used as a subunit vaccine delivery system and tuberculosis (TB) was chosen as a model disease. TB is among the deadliest infectious diseases worldwide and an effective vaccine is urgently needed. The ability of OCNs to deliver recombinant Mycobacterium tuberculosis (Mtb) proteins, Ag85B and HspX, into bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) was investigated. For immunization, OCNs were mixed with the two TB antigens as well as the adjuvant monophosphoryl lipid A (MPL). The protective efficacy was analyzed in vaccinated mice by aerosol Mtb challenge with a virulent strain of Mtb and the bacterial burdens were measured. The results showed that OCNs are highly effective in delivering Mtb proteins into the cytosol of BMDMs and BMDCs. Upon immunization, this vaccine formula induced robust Th1 immune response characterized by cytokine profiles from restimulated splenocytes and specific antibody titer. More importantly, enhanced cytotoxic $CD8^+$ T cell activation was observed. However, it did not reduce the bacteria burden in the lung and spleen from the aerosol Mtb challenge. Taken together, OCNs are highly effective in delivering subunit protein vaccine and induce robust Th1 and $CD8^+$ T cell response. This vaccine delivery system is suitable for application in settings where cell-mediated immune response is needed.

• Journal of Veterinary Science
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• 제21권2호
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• pp.22.1-22.9
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• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• 한국임상수의학회지
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• 제40권3호
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• pp.175-181
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• 2023

Fucoidan extracted from brown seaweed has a variety of biological activities. Neutrophil extracellular traps (NETs) formation is an immune response for the invasion of pathogens. Neutrophils release granule protein and chromatin that form extracellular fibers that bind microbes. These NETs degrade virulence factors and kill bacteria. The aim of this study was to investigate the effect of fucoidan on NET formation of porcine peripheral blood polymorphonuclear cells (PMNs). The NET formation was determined by fluorescence emission of propidium iodide (PI) in PMNs by a fluorescence microplate reader. The production of tumor necrosis factor (TNF)-α from peripheral blood mononuclear cells (PBMCs) was measured by ELISA method. Fucoidan itself did not show any direct effect on NET formation. However, NET formation of PMNs was increased by the culture supernatant from PBMCs treated with fucoidan. The NET formation of PMNs were also enhanced by treatment with recombinant porcine (rp) TNF-α. The ability of culture supernatant from PBMCs treated with fucoidan to increase the NET formation of PMNs was inhibited by addition of goat anti-rp TNF-α polyclonal antibody (pAb) (IgG) prior to the culture. The increase of NET formation by rp TNF-α was also inhibited by goat anti-rp TNF-α pAb (IgG). The level of TNF-α in culture supernatant from PBMCs was increased by treatment with fucoidan. These results suggest that fucoidan increases porcine NET formation, which is mediated by TNF-α produced from PBMCs.

• 한국동물위생학회지
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• 제46권4호
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• pp.263-268
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• 2023

Despite regular vaccinations, equine influenza virus (EIV) and Streptococcus equi subsp. equi (strangles) are the cause of highly contagious respiratory infections in horses. Many recent studies have reported that the concurrent administration of two vaccines could simplify horse management and minimize veterinary expenses. However, there is little information available regarding the efficacy of concurrent vaccinations against EIV and strangles. In this study, we evaluated EIV-specific antibody responses following the single EIV vaccination with the recombinant viral-vectored EIV vaccine or concurrent vaccination with the EIV and inactivated strangles vaccines. Blood samples were collected at 1-, 2-, 4-, and 8 weeks post-immunization (wpi) from each group. EIV-specific antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HAI) assay. Both single and concurrent vaccination showed similar levels of EIV-specific serum immunoglobulin g (IgG) at 1 and 2 wpi. However, at 4 to 8 wpi, the EIV-only vaccination group showed significantly higher serum IgG levels than those from the concurrently vaccinated group. The HAI titers showed similar trends as the ELISA data, except at 8 wpi when both groups presented HAI titers with no significant differences. These data demonstrate that the concurrent vaccination against EIV and strangles could compromise the humoral immune response to equine influenza between vaccination intervals, which suggests the use of the consecutive vaccination protocol for EIV and strangles rather than concurrent vaccination.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제13권2호
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• pp.134-145
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• 2010

목 적: 로타바이러스 감염역학의 변화를 연구하기 위해 A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질이 항원성이 있는지를 확인하고 이것을 항원으로 한 효소면역측정법이 로타바이러스 IgG, IgA와 IgM 항체를 정량적으로 평가할 수 있는지 확인하고자 하였다. 방 법: 경상대학교병원에서 로타바이러스 감염을 진단받은 소아들 중 진단 받기 전, 진단 당시, 회복기 이후의 연속적인 혈청을 확보할 수 있었던 22명에게서 100개의 혈청을 경상대학교병원 인체자원은행으로부터 제공받아 로타바이러스 VP6 유전자를 클로닝 하여 대장균에 발현시켜 제조한 rVP6 항원으로 한 효소면역측정법으로 IgG, IgA와 IgM 항체 역가를 측정하였다. 이 중 건강한 신생아 4명에서는 면역 블로팅을 같이 시행하였다. 결 과: 건강한 신생아와 영유아 17명에서 감염 후 확보된 혈청에서 IgG, IgA, IgM 항체 중 최소한 한 종류의 항체 역가 증가가 동반되어 있었다. 면역이 저하된 소아 5명 중 4명에서는 IgG 항체 역가는 증가되었으나 IgA 항체 역가는 2명에서만 증가하였고, IgM 항체 역가는 5명 모두 증가하지 않았다. 신생아 4명에서 시행된 면역 블로팅 검사에서는 IgM 항체인 경우는 효소면역측정법보다 예민하게 진단 초기부터 4명 모두 양성으로 판정되었다. 결 론: A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질은 항원성이 있으며 이것을 항원으로 한 효소면역측정법은 로타바이러스 감염후 IgG, IgA, IgM 항체 역가 증가를 정량적으로 평가할 수 있어 지역사회에서 발생한 로타바이러스 감염역학의 변화를 연구하는데 유용할 것으로 판단된다.

• Clinical and Experimental Pediatrics
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• 제49권8호
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• pp.857-863
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• 2006

목 적 : 의료기술과 신생아학의 발전으로 생존된 극소 저출생 체중아의 질병에 대한 관리가 더욱 필요한 시점이다. 이에 본 연구는 극소 저출생 체중아에서 영아기 B형 간염 항체 생성률과 항체 생성 실패에 미치는 요인에 대하여 알아보고자 하였다. 방 법 : 1997년 1월부터 2004년 12월까지 서울아산병원과 강릉아산병원 신생아 중환자실에 입원하였던 1,500 g 미만의 극소저출생 체중아 중에서 영아기에 B형 간염 항체에 대해 검사받은 243명을 대상으로 하였다. 영아들은 역 연령 약 40주경에 첫 간염 예방접종시행 받았으며 1개월 후, 6개월 후에 간염 예방접종을 시행 받았다. 그러나 모체의 B형 간염 항원이 양성인 13명의 영아들은 출생 시에 임신주수와 출생체중과 관계없이 B형 간염 면역글로불린과 B형 간염 예방접종을 1회 더 시행 받았다. 항체검사는 마지막 간염 예방접종 3-4개월 후에 시행하였고 B형 감염 예방접종후의 항체 양전은 항체의 역가가 ${\geq_-}10mIU/mL$로 정의하였다. 결 과 : 총 243명의 극소 저출생 체중아들의 B형 간염 예방접종 후 항체 양전율은 84.4%(205/243명)였다. 항체가 음성인 38명의 극소 저출생 체중아 중에서 재접종이 가능하였던 28명 중 17명(60.7%)에서 항체가 양성으로 전환되었다. 총 243명 중 34.6%(84/243명)을 차지한 초극소 저출생체중아의 항체 양전율은 84.5%(71/84명)이었다. 13명의 항체 음성인 초극소 저출생체중아 중 재접종이 가능하였던 10명의 항체 양전율은 80%(8/10명)로 재접종 후의 극소 저출생 체중아와 초극소 저출생 체중아의 항체 양전율은 각각 95.3%, 97.5%으로 향상되었다. 출생시 임신주수별 항체 양전율은 $28^{+0}$주 미만과 $28^{+0}-36^{+6}$주인 영아들에서 각각 83.5%(66/79), 84.8%(139/164명)이었다. 이들 중 항체가 생기지 않았던 영아에서 재접종 후 각 군에서의 항체 양전율은 각각 96%, 94.9%로 향상되었다. B형 간염 보유자에게 태어난 극소 저출생 체중아의 항체 양전율은 76.9%(10/13명)였고 모두에게서 B형 간염의 항원은 음성이였다. 항체 양전율에 영향을 미치는 요소로는 첫 B형 간염 예방접종시의 체중이 작을수록 항체 양전율이 저하됨을 알 수 있었다. 결 론 : 세 번의 B형 간염 예방접종 후 항체 생성이 되지 않은 극소 저출생 체중아들에서의 재접종은 항체 양전율을 매우 향상시킬 수 있음으로 저자들은 극소 저출생 체중아들에게 아직 국내에서 보편적으로 실시되고 있지 않는 예방접종 후 항체검사를 적극적으로 권장하는 바이다.