• Applied Biological Chemistry
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• 제51권4호
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• pp.276-280
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• 2008

식품 allergen 저감화 수단으로 자외선 조사의 타당성을 검토하고 자외선조사가 식품 단백질의 분자적 성질에 미치는 영향을 조사하고자 ovalbumin 용액에 자외선을 조사한 후 단백질의 분자량 분포와 2차구조 및 3차구조의 변화를 조사하였다. SDS-PAGE와 Gel permeation chromatography 결과 ovalbumin은 자외선 조사에 의하여 단백질이 분해되고 조사시간이 증가할수록 펩티드가 중합하는 형태를 나타냈다. Circular dichroism 연구는 자외선 조사에 의하여 ${\alpha}$-helix 구조가 감소하고 조사시간이 증가할 경우 compact denatured ovalbumin의 구조를 나타내는 2차구조의 변화를 나타냈다. 자외선 조사된 ovalbumin의 형광스펙트럼은 조사시간이 증가할수록 단백질의 maximum emission intensity가 감소하는 3차구조의 변화를 나타냈다. 결과적으로 자외선 조사는 ovalbumin의 분자적 성질을 변화시키며 allergen의 항원성을 변화시키는 수단으로 이용가능성을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.288-295
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• 2015

Salmonella enterica serovar Enteritidis is the predominant agent causing salmonellosis in chickens and other domestic animals. In an attempt to identify antigenic S. Enteritidis outer membrane proteins (OMPs) that may be useful for subunit vaccine development, we established a proteomic map and database of antigenic S. Enteritidis OMPs. In total, 351 and 301 spots respectively from S. Enteritidis strain 270 and strain 350 were detected by two-dimensional gel electrophoresis. Fifty-one antigen-reactive spots were detected by antisera on two-dimensional immunoblots and identified as 12 specific proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. OmpA and DNA starvation/stationary phase protection protein (Dps) were the most abundant proteins among the identified OMPs, comprising 22 and 12 protein species, respectively. Interestingly, we found that the Dps of S. Enteritidis is also antigenic. OmpW was also verified to have high antigenicity. These results show that OmpA, Dps, and possibly OmpW are antigenic proteins. This study provides new insights into our understanding of the immunogenic characteristics of S. Enteritidis OMPs.

• Journal of Microbiology
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• 제35권2호
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• pp.109-116
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• 1997

An extracellular proteinase of Candida albicans was purified by a combination of 0~75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45$^{\circ}C$. The addition of divalent cations, $Ca^{2+}$, Zn$^{2+}$ and $Mg^{2+}$, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe$^{2+}$, Ag$^{2+}$ and Cu$^{2+}$. With BSA as substrate, an apparent $K_m$ was determined to be 7$\times$10$^{-7}$ M and $K_i$, using pepstatin A as an inhibitor, was 8.05$\times$10$^{-8}$ M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P$_1$ position, but the enzyme activity was highly reduced when the P$_2$ position was phe or pro. This enzyme showed antigenicity against sera of patients with candidiasis.

• 한국어병학회지
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• 제24권2호
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• pp.75-84
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• 2011

우리나라 양식 넙치에서 분리한 VHSV (KR'01-1)의 glycoprotein 아미노산 배열을 기초로 단백질 전환 구조상 유연성, 친수성 및 항원성이 높고, 표면에 노출되어 있을 가능성(surface probability)이 높다고 분석되는 3개의 peptide (Gp1, Gp2, Gp3)를 선정하였다. 정제한 바이러스 입자 및 3개의 합성 peptide에 대한 polyclonal 항체를 제작하여 항원성 분석을 실시한 결과, 합성 peptide로 제작한 모든 항혈청은 Western blotting 결과 VHSV의 구조단백질과 특이적으로 결합하는 나타났으며, 이 가운데 Gp1 및 Gp2 합성 peptide에 대한 항혈청은 양식 넙치에서 분리한 VHSV를 중화시키는 것으로 나타나, peptide-based antigen의 이용 가능성을 제시하였다.

• Journal of Periodontal and Implant Science
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• 제44권1호
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• pp.33-38
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• 2014

Purpose: Autogenous bones are frequently used because of their lack of antigenicity, but good osteoconductive and osteoinductive properties. This study evaluated the biological behavior of perforated and nonperforated cortical block bone grafts. Methods: Ten nonsmoking patients who required treatment due to severe resorption of the alveolar process and subsequent implant installation were included in the study. The inclusion criteria was loss of one or more teeth; the presence of atrophy of the alveolar process with the indication of reconstruction procedures to allow rehabilitation with dental implants; and the absence of systemic disease, local infection, or inflammation. The patients were randomly divided into two groups based on whether they received a perforated (inner surface) or nonperforated graft. After a 6-month healing period, a biopsy was performed and osseointegrated implants were installed in the same procedure. Results: Fibrous connective tissue was evident at the interface in patients who received nonperforated grafts. However, full union between the graft and host bed was visible in those who had received a perforated graft. Conclusions: We found that cortical inner side perforations at donor sites increased the surface area and opened the medullary cavity. Our results indicate an increased rate of graft incorporation in patients who received such perforated grafts.

• Parasites, Hosts and Diseases
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• 제32권4호
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• pp.243-248
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• 1994

질편모충 항원 분석의 일환으로 막항원의 분석을 시도하였다 수확 세척된 질편모충 HY-l의 homogenate를 sucrose step-gradient를 이용하여 differential centrifugation하였으며 25%/45%의 sucrose 경계면으로녁터 막분회을 얻었다. 분리된 막분회은 transmission electron microscopy를 통하여 순수 분리되었는지 확인하였고 효소면역 전기영동 이적법(EITB)을 이용하여 항원성을 관찰하였으며 그 결과는 다음과 같았다. 분리된 막분획은 투과전자 현미경상에서 extended sheet나 concentric vesicle의 형태가 거의 균질하게 분포하고 있었으며 막 분획에서 특징적으로 나타나는 trilaminal appearance를 보여 질편모충의 막분획이 순수 분리된 것으로 간주할 수 있었다. 분리된 막분획은 EITB상에 토끼의 항혈청과 반응하였을 때 46, 60, 110, 120, 130 및 150 kfDa에서 항원성이 있는 반응대가 관찰되었으며 N-hydroxysuccinimido-biotic으로 표지하여 분리된 표면항원의 녁획과 비교하였을 때 60 KDa의 항원 분획이 서로 일치하는 것으로 나타났다. 따라서 60 kfDa 의 항원 분획은 료면 항원임을 확신할 수 있었다.

• Parasites, Hosts and Diseases
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• 제51권5호
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• pp.503-510
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• 2013

Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7395-7399
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• 2014

Background: Persistent human papillomavirus (HPV) infection, especially with high-risk types such as HPV16 and HPV18, has been identified as the primary cause of cervical cancer. E6 and E7 are the major onco-proteins of high-risk HPVs, which are consistently expressed in HPV infected tissues but absent in normal tissues and represent ideal therapeutic targets for immunotherapy of cervical cancer. Materials and Methods: In this study, the optimized fusion gene HPV18 E6E7 (HPV18 ofE6E7) was constructed according to genetic codon usage for human genes. At the same time, for safety future clinical application, a mutant of HPV18 ofE6E7 fusion gene was generated by site-directed mutagenesis at L52G for the E6 protein and C98G for the E7 protein. Results: HPV18-E6E7 mutant (HPV18 ofmE6E7) constructed in this work not only lost the transformation capability for NIH 3T3 cells and tumorigenicity in BALB/c nude mice, but also maintained very good stability and antigenicity. Conclusion: These results suggest that the mutant should undergo further study for application as a safe antigenspecific therapeutic vaccine for HPV18-associated tumors.

• 대한바이러스학회지
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• 제27권2호
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• pp.151-160
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• 1997

In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-4T-3, a GST gene fusion vector, yielding $pGCV_{pol}$. This construct expressed a Snow Mountain-like HuCV replicase under the control of the IPTG-inducible tac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing $pGCV_{pol}$ products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.

• IMMUNE NETWORK
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• 제6권3호
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• pp.145-153
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• 2006

Background: Tumor cell lysate has been considered as a preferential antigen source for the therapeutic dendritic cell pulsing. Our experiences with in vivo study with animal tumor model indicate the tumor cell lysate dependent differential effect of DC therapy. Our previous data show that MC38 lysate pulsed-DC induced stronger ag-specific immunity than CT26 lysate pulsed-DC in vitro. In this study we tried to reveal the mechanism for differential induction of ag-specific immunity of different colon cancer cell lysate pulsed-DCs. Methods: MC38 and CT26 cell lines were prepared as lysate by freezing-thawing procedure. Tumor cell antigenicity was confirmed by detecting the surface expression of MHC I/II & B7.1/2 molecules. IL-10, IL-12 and TGF-beta in the tumor cell lysate were detected by ELISA and the presence of heat shock proteins were analysed by western blotting. Results: The secretion of IL-10, a immune-inhibitory cytokine was about 470% higher in CT26 lysate than in MC38. Hsp 70 was detected only in the MC38 lysate but not in the CT26. On the other hand, Hsp 60 and 90 expression were not different in two colon cancer cell lysates. Conclusion: In two different colon cancer cell lysate, immune inhibitory IL-10 (higher in CT26) and Hsp70 (MC38 superiority) were differentially expressed. These data indicate that higher agspecific immunity induction by MC38 lysate pulsed-DC may due to the expression of hsp70 and lower secretion of IL-10, a immune-inhibitory cytokine than CT26 lysate. The significance of other cytokine and the surface marker expression will be discussed.