• 한국어병학회지
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• 제11권1호
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• pp.43-50
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• 1998

우리 나라의 양식 해산어에서 분리된 종양을 유발하는 iridovirus가 폐사를 유발하는 iridovirus와 같은 종류인지를 확인하기 위하여 면역학적 특성을 비교하여 보았다. SDS-PAGE를 통하여 이들 바이러스의 구조단백질을 비교한 결과 종양을 유발하는 iridovirus와 폐사를 유발하는 iridovirus는 서로 다른 크기의 단백질을 소유하는 것으로 확인되었다. 종양을 유발하는 iridovirus에 대한 단일클론항체를 사용한 Western blotting실험을 통하여 구조 단백질의 항원성을 조사한 결과 종양을 유발하는 iridovirus 의 경우 분자량 150 kDa의 구조단백질이 면역 유도 특성이 있음이 확인되었다. 반면에 폐사를 유발하는 iridovirus는 종양을 유발하는 iridovirus에 대한 단일클론항체들과는 전혀 반응을 하지 않았다. 이 결과로부터 종양을 유발하는 iridovirus와 폐사를 유발하는 iridovirus의 구조단백질들은 서로 다른 항원성을 지님을 알 수 있었으며 이는 두 iridovirus들이 서로 다른 type일 가능성이 높음은 나타내고 있다.

• 생명과학회지
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• 제20권12호
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• pp.1743-1749
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• 2010

Edwardsiella tarda는 그람 음성의 장내세균과의 주요 어병세균으로 어류에 edwardsiellosis를 유발하는 전신감염성 병원체이다. 최근 병원성 세균의 외막 단백질들은 세균성 감염에 있어서 숙주와 반응하여 면역반응을 유도하는 것으로 여겨져 연구가 되고 있다. 일본의 연구팀은 어류에서 에드워드병의 원인체인 E. tarda의 37 kDa 단백질이 넙치에서 높은 항원성을 제시하는 것을 보고하였다. 또한 그 연구자들은 37 kDa 단백질의 N-말단 아미노산 서열이 GAPDH와 대응하는 것을 밝혔다. 본 연구에서는 다른 세균에서 알려진 N-말단 서열을 기반으로 primer를 제작하여 이에 상응하는 E. tarda DNA를 증폭하고 클로닝하였다. 이 DNA단편의 염기서열은 예상한 바와 같이 세균의 GAPDH유전자인 gapA와 높은 상동성이 있고, E. tarda GAPDH (etGAPDH)의 아미노산 서열은 다른 장내세균의 GAPDH와 70% 이상의 상동성을 보이는 것을 확인하였다. E. tarda의 외막단백질에 특이적으로 반응하는 항체를 이용하여 E. tarda의 GAPDH가 외막에 존재한다는 것을 증명하였고, gapA의 염기서열을 바탕으로하여 재조합 GAPDH를 과발현 시켰다. 과발현된 재조합단백질 GAPDH는 GAPDH 특이적인 항체를 제조하는데 사용되었고, 또한 넙치에 면역시켜 단일 단백질 백신으로서의 활용도를 모색하였다. 비록 재조합 GAPDH가 면역된 넙치에서 GAPDH에 특이적인 항체가 증가하였음에도 불구하고, E. tarda로 공격실험을 하였을 때 면역된 넙치의 생존율이 12.5%로 측정되어 면역된 그룹과 면역되지 않은 그룹간에 큰 차이가 없는 것이 확인되었다.

• Parasites, Hosts and Diseases
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• 제30권3호
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• pp.209-218
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• 1992

혈청이나 뇌척수액에서 특이항체를 증명하여 유구낭미충증을 진단함에 있어 유구낭미충의 낭액이 가장 민감하고도 특이한 항원임은 이미 알려졌다. 낭액을 항원으로 사용하여도 다른 기생충증과 빈도는 낮지만 교차반응이 발생하므로 교차반응을 일으키는 구성단백질에 대하여 더 관찰할 필요가 있다. 이 연구에서는 유구낭미충 낭액을 Sephacryl S-300 Superfine 젤로 분획하고, 낭액 및 그 분획이 낭액을 면역시켜 만든 토끼 항혈청 (RACF)과 반응하는 양상을 면역전기영동법으로 관찰하여 낭액의 구성 단백질의 성질을 분석하고자 하였다. 유구낭미충 장액을 젤여과법으로 분획하면 제 3분회과 제 4분회에 단백질이 가장 많이 포함되었다. 제 3분획은 disc-PAGE상 분자량 150 kDa인 것으로 이미 알려진 Band C가 주(주) 구성 단백질이었다. 제 4분획은 분자량이 약 65kDa일 것으로 추정되는 Band N 단백질이 주 구성단백질이었다. 낭액에 대한 토끼 항혈청을 낭액과 면역전기영동법으로 반응시키면 주 침강대 2개와 작은 침강대 7∼8개를 형성하였는데 낭액의 제 3분획은 주 침강대 중 밖에 위치한 긴 침강대를 형성한 반면, 제4분획은 주 침강대 중 안쪽으로 위치한 짧은 침강대를 형성하였다. 유구낭미충과 혈청학적 교차반응을 가장 많이 일으키는 포충(포충, hydatid cyst)의 낭액과 RACF(유구낭미충 낭액에 대한 토끼 항혈청)을 면역전기영동법으로 반응시킨 바, 제 4분획이 반응한 위치에서 침강대를 형성하여 제 4분획의 주구성 단백질이 포충증과의 혈청학적 교차반응의 원인인 것으로 추정하였다. 유구낭미충증으로 이미 진단한 환자의 혈청 중 효소면역 측정 법으로 항체가가 매우 높았던 혈청 8개를 선택하여 유구낭미충 낭액 및 유구낭미충 충체 추출액과 각각 면역전기영동법으로 반응시킨 바 각각 7명에서 침강대를 형성하였고, 제 3분회 및 제 4분회으로 면역전기영동법으로 반응시킨 환자혈청 4개 중에서는 제 3분획에서는 4개 모두가 침강대를 형성한 반면 제 4분 회에서는 침강대가 혈청 2개에서 형성되었다.

• Archives of Pharmacal Research
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• 제22권5호
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• pp.485-490
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• 1999

The mechanism of action of fish oil (FO), currently used in different chronic inflammatory conditions such as rheumatoid arthritis (RA), is not completely understood, although it is thought that it could alter the metabolism of endogenous autacoids. In addition, we hypothesized that the known capability of fatty acids (FA) of stabilizing serum albumin and perhaps other proteins, may be of pharmacological relevance considering that it is shared by other anti-rheumatic agents (e.g. nonsteroidal antiinflammatory drugs). Thus, we studied the effect of oral administration of FO and corn oil (CO), a vegetable oil with a different composition, on the stability of rat serum proteins, evaluated buy a classical in vitro method based on heat-induced protein denaturation. FO, and, to a lower extent, CO inhibited heat-induced denaturation of rat serum (RS): based on the inhibitory activity (EC50) of the major fatty acids against heat-induced denaturation of RS in vitro, it was possible to speculate the in vivo effects of palmitic acid (C16:0) and eicosapentaenoic acid (EPA, C20:5, n-3) may be more relevant than that of linolenic acid (C18:2). To better investigate this phenomenon, we extracted albumin from the serum of animals treated or not with FO with a one-step affinity chromatography technique, obtaining high purity rat serum albumin preparations (RSA-CTRL and RSA-FO), as judged by SDS-PAGE with Coomassie blue staining. When these RSA preparations were heated at $70^{\circ}C$ for 30 min, it was noted that RSA-FO was much more stable than RSA-CTRL, presumably due to higher number of long chain fatty acids (FA) such as palmitic acid or EPA. In conclusion, we provided evidences that oral administration of FO in the rat stabilizes serum albumin, due to an increase in the number of protein bound long chain fatty acids (e.g. palitic acid and EPA). We speculate that the stabilization of serum albumin and perhaps other proteins could prevent changes of antigenicity due to protein denaturation and glycosylation, which may trigger pathological autoimmune responses, suggesting that this action may be involved in the mode of action of FO in RA and other chronic inflammatory diseases.

• Biomolecules & Therapeutics
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• 제14권1호
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• pp.30-35
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• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

• 미생물학회지
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• 제42권4호
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• pp.294-298
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• 2006

Streptomyces somaliensis가 생산하는 phospholipase D (PLD)를 정제하기 위하여 펩티드 항체 결합 면역 친화 크로마토그래피용 칼럼을 개발하였다. 단백질 구조 예측 프로그램과 Streptomyces PLD X-선 결정구조를 참조하여, S. somaliensis PLD의 1차 구조로부터 항원특성이 높고. 표면에 위치하는 것으로 예상된 5종류의 펩티드들을 epitope로 선정한 뒤, 이에 대한 항체로 면역친화 크로마토그래피용 칼럼을 제작하였다. 배양 농축액을 칼럼에 통과시켜 정제한 활성 분획을 SDS-PAGE 및 Western blot 결과, 칼럼 종류에 따라 순수한 PLD또는 35 kDa의 단백질 불순물만을 포함하는 PLD 정제 분획을 보여 면역친화 칼럼의 높은 항원결합 특이성을 보여주었다. 그러나 수용액상에서 PLD 자체의 구조적 불안정성 때문에 정제 후 PLD의 특이적 활성 및 정제 수율은 낮았다.

• 한국동물위생학회지
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• 제34권2호
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• pp.103-109
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• 2011

Asia1/Shamir that has been recommended by World Reference Laboratory for foot-and-mouth disease (FMD) is used as a vaccine strain, and is being prepared in many countries including Korea. Although it is assumed that vaccine strain Asia1/Shamir has a wide antigenicity, sufficient molecular biological analysis has not been accomplished yet. Complete genome sequence analysis showed that the region with the most severe variations was 1D region of structural protein-coding sequence; particularly amino acid 141~157 residues in 1D region RGD sites for binding to susceptible cells. In addition, five amino acids in 1D region were identified as characteristic sites that are different from other known Asia1 viruses. Asia1/Shamir strain was shown to be genetically similar to group VI that had occurred in the Middle East, but showed low level of genetic similarity to the group V viruses that had occurred in the Southeast Asia and China. It is considered that, if these viruses, group I and II including group V are introduced into Korea, care would be paid in case of inoculating the vaccine strain Shamir available in Korea.

• 한국동물위생학회지
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• 제15권1호
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• pp.32-45
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• 1992

In order to establish and enzyme-linked immunosorbent assay to ILTV, field virus strain of ILTV was propagated in chorioallantoic membrane of the embryonated eggs. purified and used as antigen. The antisera selected from the field samples and immunized chickens based on serum neutralization test were used as the standard positive and negative sera in all tests. It was found that optimal antigen concentration was $2{\mu}g$ of protein per well and a 1 : 100 dilution of standard serum showed low background optical density with negative serum and high P/N values of positive sera. A 1 : 500 dilution of the rabbit anti-chicken IgG peroxidase conjugate produced a high P/N values and thirty minutes was chosen as suitable time to read the optical density of the enzyme substrate reaction and optical density was consistent during the 16 hours after stopper was treated. When coated antigen was kept on microplate for varying time up to 16 hours at $4^{\circ}C$ or $37^{\circ}C,$ no significant difference was observed between the treatment. The coated antigen could be kept without change of antigenicity for at least one month at $-70^{\circ}C,\; -20^{\circ}C,\; 4^{\circ}C$ and room temperature. When blocking buffer contanining bovine serum albumin was mixed directly with conjugate and serum at 10% level induced higher P/N values compared to blocking antigen coated microplate with the same blocking buffer. The coefficience of correlation between ELISA and SN test was 0.577. When antibody response of chickens, vaccinated with ILTV, was examined by ELISA and SN test, antibody rising and decay pattern between the two test was similar until 11 weeks of age. However 12 weeks onward antibody titer checked on by SN test was slightly lower than that tested by ELISA.

• Clinical and Experimental Vaccine Research
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• 제12권2호
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• pp.156-171
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• 2023

Purpose: The development of vaccines that confer protection against multiple avian influenza A (AIA) virus strains is necessary to prevent the emergence of highly infectious strains that may result in more severe outbreaks. Thus, this study applied reverse vaccinology approach in strategically constructing messenger RNA (mRNA) vaccine construct against avian influenza A (mVAIA) to induce cross-protection while targeting diverse AIA virulence factors. Materials and Methods: Immunoinformatics tools and databases were utilized to identify conserved experimentally validated AIA epitopes. CD8⁺ epitopes were docked with dominant chicken major histocompatibility complexes (MHCs) to evaluate complex formation. Conserved epitopes were adjoined in the optimized mVAIA sequence for efficient expression in Gallus gallus. Signal sequence for targeted secretory expression was included. Physicochemical properties, antigenicity, toxicity, and potential cross-reactivity were assessed. The tertiary structure of its protein sequence was modeled and validated in silico to investigate the accessibility of adjoined B-cell epitope. Potential immune responses were also simulated in C-ImmSim. Results: Eighteen experimentally validated epitopes were found conserved (Shannon index <2.0) in the study. These include one B-cell (SLLTEVETPIRNEWGCR) and 17 CD8⁺ epitopes, adjoined in a single mRNA construct. The CD8⁺ epitopes docked favorably with MHC peptidebinding groove, which were further supported by the acceptable ∆G_bind (-28.45 to -40.59 kJ/mol) and Kd (<1.00) values. The incorporated Sec/SPI (secretory/signal peptidase I) cleavage site was also recognized with a high probability (0.964814). Adjoined B-cell epitope was found within the disordered and accessible regions of the vaccine. Immune simulation results projected cytokine production, lymphocyte activation, and memory cell generation after the 1st dose of mVAIA. Conclusion: Results suggest that mVAIA possesses stability, safety, and immunogenicity. In vitro and in vivo confirmation in subsequent studies are anticipated.

• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1717-1721
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• 2008

Severe acute respiratory syndrome (SARS) is a life-threatening emerging respiratory disease caused by the coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is highly antigenic and may be a suitable candidate for diagnostic applications. We constructed truncated recombinant N proteins (N1 [1-422 aa], N2 [1-109 aa], and N3 [110-422 aa]) and determined their antigenicity by Western blotting using convalescent SARS serum. The recombinants containing N1 and N3 reacted with convalescent SARS serum in Western blotting. However, the recombinant with N2 did not. In ELISA using N1 or N3 as the antigens, positive results were observed in 10 of to (100%) SARS-CoV-positive human sera. None of 50 healthy sera gave positive results in either assay. These data indicate that the ELISA using N1 or N3 has high sensitivity and specificity. These results suggest that the middle or C-terminal region of the SARS N protein is important for eliciting antibodies against SARS-CoV during the immune response, and ELISA reactions using N1 or N3 may be a valuable tool for SARS diagnosis.