Analysis of proteome in clonorchis sinensis adult worms and identification of antigenic protein by immuno-proteomics

이명노 Graduate School, Korea University 2013 국내박사

Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. I identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, I found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. These proteins will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies.

Molecular characters and induction profiles of HIF-1α and other bHLH-PAS domain-containing proteins identified in a carcinogenic liver fluke Clonorchis sinensis

오규석 가천대학교 대학원 2019 국내석사

Clonorchis sinensis, a trematode parasite that invades the hepatobiliary tracts of vertebrate hosts with limited available oxygen requires a significant amount of oxygen for its sexual reproduction and energy metabolism. However, little is known about molecular mechanisms involved in the maintenance of oxygen homeostasis and/or adaptation to the hypoxic environments in the liver fluke. In this study, we characterized molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix-loop-helix and Per-Arnt-Sim (bHLH-PAS) domain-containing proteins such as HIF-1β, single-minded protein (SIM), and aryl hydrocarbon receptor (AHR), which may prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed functional domains that are tightly conserved in each of the bHLH-PAS subfamilies, whilst the oxygen-dependent degradation domains could not be defined in the probably partial C. sinensis HIF-1α (CsHIF-1α) protein. Expression of the CsHIF-1α gene was highly induced in worms either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite also affected the CsHIF-1α expression depending on the surrounding oxygen tension. Treatment of a prolyl hydroxylase inhibitor under normoxia condition resulted in an increase of the CsHIF-1α level. Conversely, the other bHLH-PAS genes were less responsive to these exogenous stimuli. Taken together, our data suggest that the HIF-PHD-VHL triad constitutes a key regulator of physiological responses to hypoxia also in trematodes including C. sinensis. To our best knowledge, this is the first report concerning identification and characterization of HIF-1α and relevant proteins in the phylum Platyhelminthes. Clonorchis sinensis는 제한된 이용 가능한 산소로 척추 동물 숙주의 간 담관을 침범하는 흡충류 기생충으로 유성생식과 에너지 대사에 상당한 양의 산소가 필요하다. 그러나 산소 항상성의 유지 또는 담관 내에서와 같은 저산소 환경에서의 적응에 관련된 분자 기전에 대해서는 알려진 바가 거의 없다. 본 연구에서는, hypoxia-inducible factor-1α (HIF-1α)와 basic helix-loop-helix 및 Per-Arnt-Sim (bHLH-PAS) 도메인을 포함하는 단백질인 HIF-1β, single-minded protein(SIM) 및 aryl hydrocarbon receptor (AHR) 등의 분자 구조와 유도 특성을 확인하였고, 이들은 C.sinensis에서 저산소증에 대한 적응을 돕는 것으로 확인되었다. 이들 단백질은 bHLH-PAS 서브 패밀리 각각에서 강하게 보존되는 기능성 도메인을 보유하고 있는 반면, C.sinensis HIF-1α (CsHIF-1α) 단백질에서는 산소 의존성 분해 도메인을 부분적으로 규명 할 수 없었다. CsHIF-1α 유전자의 발현은 저산소 상태에 노출되거나 인간 담관 세포와 함께 배양 된 간흡충에서 높은 정도로 유도되었다. 산소 이외에 산화 질소와 아질산염 또한 주변 산소 분압에 따라 CsHIF-1α 발현에 영향을 주었다. 정상 산소 상태에서 수산화억제제(prolyl-4-hydroxylase inhibitor)를 처리한 경우에는 CsHIF-1α 발현이 증가하였다. 반면에 다른 bHLH-PAS 유전자들의 경우, 이러한 외부 요인들에 대해 비교적 덜 반응하였다. 결론적으로, 본 연구에서는 C. sinensis를 포함한 흡충류에서도 HIF-PHD-VHL triad가 저산소증에 대한 생리적 반응의 중요한 조절자라는 사실을 확인하였다. 이 연구는 편형동물에서 HIF-1α 및 관련 단백질의 동정 및 특성을 규명한 최초의 연구이다.

Blanching에 의한 햇순나물의 이화학적 특성 및 기능성 변화

김민하 영남대학교 대학원 2012 국내석사

Clonorchis sinensis infestation induces 3-dimensional invasion of cholangiocarcinoma

Jihee Won 고려대학교 대학원 2014 국내석사

Clonorchis sinensis (C. sienensis) is a worm parasite that is prevalent in East Asian countries, and it causes chlonorchiasis, diverse symptoms occurred by infection of C. sinensis. Since epidemiological correlation between C. siensnsis infestation and CCA was reported, initiation of CCA involved in C. sinensis has been researched in many experimental research cases such as current studies suggesting that excretory-secretory products (ESPs), molecules that are secreted by this parasite and released into surround hepatobiliary tissue, play important role in host-parasite interaction related to CCA initiation, whereas progress of CCA has not been. Now, I investigated the possibility that C. sinensis infestation affects on poor prognosis of CCA by inducing the invasion of CCA. In the CCA cells with ESPs treatment, expression of focal adhesion proteins and secretion of matrix metalloproteinases (MMPs), related to mobility of the cells, were increased. Furthermore, Using an advanced CCA culture models based on a novel microfluidic assay, I quantitatively addressed the role of ESPs gradients from C. sinensis in promoting the invasion of CCA cells into neighboring ECM.

Purification and preliminary crystallization of 14-3-3 protein from clonorchis sinensis

조용욱 Graduate School, Korea University 2013 국내석사

Clonorchis sinensis 14-3-3 proteins (C.sin14) are considered the antigenic proteins of C. sinensis which is widespread in Asian countries, including the Repbulic of Korea and China. The consumption of raw freshwater fishes is one of the major factors for C.sinensis infection. Since the infection of C. sinensis can affect people’s health in several ways, diagnostic methods for the C. sinensis infection are needed to prevent serious health problems, such as the development of cholangiocarcinoma. In this study, due to the possibility that the C.sin14 can be tagetted for a vaccine, we attempted to determine the C.sin14 structure. The recombinant 14-3-3 protein from C. sinensis was overexpressed, purified and crystallized. The C.sin14 purified by protein purification methods including chromatographic techniques was used for protein crystallization. For the primary step to determine the structure of C.sin14, I performed the crystallization by using hang-drop vapour diffusion method. I had grown the appropriate crystals at two optimal conditions. By using precipitant 1.10 M Ammonium tartrate (pH 7.0), the appropriate crystals were formed and the optimal concentration of the C.sin14 was about 20 mg/ml. The crystal grew to size about 0.2 mm x 0.2 mm x 0.1 mm within one week. The second condition is 0.14 M sodium fluoride, as a precipitant 20 % (w/v) polyethyleneglycol 3,350 (pH 7.3). The size is about 0.1 mm x 0.2 mm x 0.1 mm within two weeks.. A diffraction data set was collected to 3.0 Å resolution by using synchrotron radiation at photon factory BL1A beam line in Japan. The crystals of C.sin14 belong to the space group P432, P4132, P4332 or P4232 and a solvent content is 54.6%. The crystals belong to the cubic space group, with unit-cell parameters a=b=c=221.922 Å, and α=β=γ=90°. The asymmetric unit contains 6 molecules, corresponding to a Matthews coefficient (VM) Value of 2.71 Å3 Da-1. Determination of the protein structure is currently in progress.

Establishment, expression and characterization of Miscanthus sinensis transformed by Agrobacterium tumefaciens using COMT gene related to lignin biosynthesis : 리그닌 생합성 관련 유전자 COMT 발현특성 구명 및 Agrobacterium을 이용한 참억새 형질전환체계 확립

JiHyeYoo 강원대학교 일반대학원 2016 국내박사

Miscanthus sinensis is a diploid hybrid and a temperate, perennial, cross-pollinating grass used as bioenergy plant, biomass production and high quality cellulose and ethanol production. The establishment of EST data base is important to study classification and function of gene groups in Miscanthus sinensis. A cDNA library from Miscanthus after UV-B treatment was prepared from mature mRNAs, and 1,000 cDNA clones were partially sequenced. A total of 827 unigenes, including 72 contigs and 755 singletons, were identified after assembly of the expressed sequence tags (ESTs). To analyze the ESTs from the UV-induced cDNA library, grouped the sequences and classifications based on the best homology match of BLASTX searches against various plant protein sequences. The functional classification sequences belonged to 15 categories including metabolism, energy, cell cycle, transcription, protein synthesis, protein fate, protein with binding function, regulation of metabolism and protein function, cell transport, signal transduction, cell defense, interaction with the environment, cell fate, biogenesis of cellular components, and subcellular localization, which were considered to be of interest for UV-response signaling. Firstly, I isolated the caffeic acid O-methyl-transferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, I produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidase expression, which are related to the phenyl-propanoid pathway and antioxidant activity. The MsCOMT down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT down-regulated transgenic plants. I also observed a morphological difference with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway. This study was carried out to enhance the transformation rate from Miscanthus sinensis, to characterize the agronomic and morphological traits, the expression of MsCOMT in transgenic plant, the variation in lignocellulosic component, and the variation in phenolic compounds content. An efficient transformation of callus from M. sinensis was established using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish stable transformation system, I obtained high transformation rate from callus by various transformation factor explant type, strain, co-culture periods, acetosyringone concentration, and selective marker. Finally, in this study, seven putative transgenic plants were obtained. Through various tests including PCR analysis and southern blot were to detect antisense of COMT digested Xba I and Sac I restriction enzymes. The biomass of the control plant was superior than transgenic plants. Total lignin content of transgenic plants was lower than the control plant, due to the reducing of COMT gene expression related to lignin production. Cellulose and hemicellulose contents in transgenic plants were not increased. Variations in cellulose and hemicellulose content had no correlation with variations in lignin content of transgenic plants. Total phenolic compounds content was generally lower in the stem of transgenic plants than in the leaf. In the control plant, the ratio of phenolic compounds content in leaves and stems was almost similar. 참억새(Miscanthus sinensis)는 국내에 흔히 자생하는 다년생 C4 식물이며, 비농경지 지역에 서식하는 영년생 잡초로 알려져 있으나, 건물수량이 ha당 20 ~ 40톤 정도로 매우 높아 바이오에탄올 생산을 위한 원료작물로 활용하고자 하는 연구가 활발히 이루어지고 있다. 그러나 참억새의 높은 리그닌 함량은 바이오에탄올로의 전환효율을 감소시켜 실용화에 장벽이 되고 있다. 따라서 본 연구는 리그닌 생합성 관련 유전자인 COMT (Caffeic acid O-methyl-transferase)의 발현특성을 구명하고 Agrobacterium을 이용하여 참억새의 형질전환체계를 확립하고, 형질전환체의 생육특성 및 성분분석, 리그닌 함량의 변화를 확인하였다. UV를 처리한 참억새로부터 mRNA를 분리하여 cDNA library를 제작하였으며, EST 분석 결과 metabolism 관련 유전자 35개 에너지 관련 유전자 24개 세포주기 및 DNA 과정 관련 유전자 4개, transcripton 관련 유전자 14개, 단백질 합성 관련 유전자 21개, Protein fate 관련 유전자 54개, 공통인자 및 연결 기능 단백질 관련 유전자 223개, 단백질 기능 조절 관련 유전자 29개, 세포상호작용 시그널 관련 유전자 7개, 식물방어작용 관련 유전자 24개 등으로 유전자들의 기능을 분류하였다. 이중 리그닌 및 에너지 관련 대사 유전자들을 유용유전자로 선별하였고 MsCOMT로 명명하였다. 참억새 내에 있는 COMT 유전자의 full length 염기서열을 얻었고 NCBI blast를 이용하여 타작물의 COMT와 유사성을 분석하였다. COMT를 down regulation 시켜 유전자의 기능을 억제하여 벡터를 제작하였고, 클로닝된 MsCOMT 유전자를 이용하여 모델식물인 담배와 참억새에 형질전환을 시도하였으며, 그 체계를 확립하여 COMT 유전자의 발현 및 기능을 구명하였다. 형질전환 체계 확립은 식물체부위, strain, 공동배양 기간, acetosyringone 농도, 선발마커 등 다양한 요인을 적용하여 연구하였다. 미숙화기와 종자에 Agrobacterium strain LBA4404를 접종 한 후 3~4일간 공동배양할 시 캘러스 형성율 및 재분화율이 증가하였으며, kanamycine 선발마커와 aceto-syringone 200 uM에서 재분화율이 증가하였고, 참억새 형질전환체는 7개체를 얻었다. 형질전환체 참억새의 초장 및 줄기 직경, 엽장, 엽폭, 잎 세포 크기 등이 대조구에 비해서 작은 형태적 특징을 나타냈다. 형태적으로 왜소한 형질전환체는 페놀 및 플라보노이드 함량도 대조구에 비해 낮았으며, 이는 모델식물인 담배에서도 비슷한 결과를 나타냈다. 모델식물 담배 형질전환체에서 MsCOMT-AS 유전자가 발현할수록 리그닌 생합성에 관여하는 유전자인 APX, PAL의 발현량이 줄어드는 것을 확인하였다. MsCOMT-AS 유전자 기능으로 인하여 참억새 형질전환체에서 리그닌의 함량이 감소하는 것을 확인하였으며, 줄기와 잎 부분에서 대조구에 비해 약 50% 정도 감소되는 것을 확인할 수 있었다. 반면에 셀룰로스 및 헤미셀룰로스의 함량은 대조구와 차이가 없는 것을 확인하였다. 연구결과 COMT 유전자는 리그닌 합성에만 직접적인 영향이 있는 것으로 사료되며, 셀룰로스와 헤미셀룰로스 합성에는 관여 정도가 적은 것으로 사료된다.

Anti-inflammatory effects of ethanolic extracts of Chaenomeles sinensis leaves : 모과잎 에탄올 추출물의 항염증 효과

한영기 건국대학교 일반대학원 2015 국내석사

The Chaenomeles sinensis fruit is commonly used to treat conditions such as pneumonia and bronchitis, but a dearth of research on the leaf exists. In this study, we investigated the anti-inflammatory effects of C. sinensis leaf extract (CSL) on LPS-stimulated RAW 264.7 cells. The 70 % ethanol extract of CSL (CSLE) significantly inhibited NO, IL-6, and TNF-α production; additionally, the CSLE suppressed LPS-stimulated inducible NOS (iNOS), IL-1β, and phospho-STAT1 expression. In this study, we investigated whether CSLE decreases the production of inflammatory mediators through the inhibition of the TRIF-dependent pathways. Our results suggest that CSLE attenuates LPS-stimulated inflammatory responses in macrophages via the regulation of the TLR4 pathways, providing scientific support for its traditional use in the treatment of a variety of inflammatory diseases. Chaenomeles sinensis는 일반적으로 폐렴, 기관지염 등에 사용된다. 본 연구에서는 lipopolysaccharide로 자극시킨 RAW 264.7세포에서 Chaenomeles sinensis 잎 추출물의 항 염증 효과를 조사하였다. 그 결과 Chaenomeles sinensis 잎 70% 에탄올 추출물이 NO 및 iNOS 생성을 억제 시켰다. 또한 Chaenomeles sinensis 잎 70% 에탄올 추출물은 IFN-β 및 인산화된 STAT1의 생성을 억제 시켰다. 위 결과를 바탕으로 TRIF-dependent 경로의 염증성 매개체 생성 억제 확인실험을 하였다. 실험 결과 Chaenomeles sinensis 잎 70% 에탄올 추출물은 LPS로 자극되어 생성된 염증성 사이토카인인 TNF-α, IL-6 및 IL-1β를 억제 시켰다. 연구 결과를 종합하면 Chaenomeles sinensis 잎 70% 에탄올 추출은 LPS로 자극된 반응물들을 감소시키며, 염증 메커니즘을 조절 할 수 있는 가능성을 과학적인 증거로 제시한다. 또한 이 연구를 바탕으로 염증성 질병에 치료제로써 사용할 가치가 있다고 보여진다.

간흡충 항원 유전자의 클로닝 및 재조합 항원을 이용한 혈청학적 진단

심서보 연세대학교 대학원 2002 국내석사

토끼의 담도로부터 얻은 살아 있는 간흡충을 protease inhibitor cocktail이 들어 있는 RPMI배지에 넣고 유지하여 분비배설항원을 얻었다. 이 분비배설항원 중 21 kDa 부위의 단백질을 면역시킨 흰쥐의 혈청과 간흡충증환자 혈청으로 간흡충 성충의 cDNA library를 immunoscreening하여 강하게 반응하는 양성 clone을 선별해 내었다. 간흡충증 환자 혈청을 이용해 분리한 2개의 clone중 하나는 9개의 amino acids가 23회 반복되는 구조를 포함하는 265개의 amino acids로 구성되었고 glycine의 비중이 높아 GRCSP2라 명명하였다(GenBank Accession number AF461709). 다른 하나는 10개의 amino acids가 22회 반복되는 구조를 포함하는 269개의 amino acids로 구성되었고 proline의 비중이 높아 PRCSP라 명명하였다(GenBank Accession number AF461711). 간흡충 분비배설항원을 면역시킨 흰쥐의 혈청을 이용해 분리한 4개의 clone은 모두 흡충류의 myoglobin과 유사성이 높아 C. sinensis myoglobin이라 하였고 이는 150개의 amino acids로 구성 되었다(GenBank Accession number AF461710). GRCSP2와 PRCSP는 간흡충 total RNA 상에서 모두 1.05 kb 크기로 발현되었고 C. sinensis myoglobin은 0.57 kb 크기로 발현되었다. Southern blotting 결과 3가지의 유전자 모두 간흡충 genomic DNA에 존재함을 확인하였다. GRCSP2와 PRCSP의 recombinant protein을 purification하여 간흡충증의 진단에의 민감도를 Western blotting으로 검사해 본 결과 각각 93.3%와 86.7%를 나타냈으며 폐흡충증, 요코가와흡충증, 회충증, 스파르가눔증 환자 혈청에 교차반응을 보였고, 특히 PRCSP가 더 많은 교차반응을 나타냄을 확인하였다. Clonorchis sinensis adult worms obtained from the biliary tract of an experimentally infected rabbit were suspended in RPMI including a protease inhibitor cocktail in order to obtain C. sinensis excretory-secretory(ES) antigens. A C. sinensis cDNA library was screened with immune rat sera which were immunized with the 21 kDa protein of ES antigens and also screened with C. sinensis-infected human sera. Two clones were isolated by using C. sinensis-infected human sera. The cDNA insert of one clone contained a single open reading frame(ORF) of 795 base pairs encoding for 265 amino acids including 23 tandem repeats of 9 amino acids. Because glycine was a major component in its content, the protein was named GRCSP2(GenBank Accession number AF 461709). The cDNA insert of the other contained a single ORF of 807 base pairs encoding for 269 amino acids including 22 tandem repeats of 10 amino acids. Because proline was a major component in its content, the protein was named PRCSP(GenBank Accession number AF 461711). Four clones were isolated by using the immune rat sera against 21 kDa protein of ES antigens. The cDNA inserts of four clones were very similar and contained a single ORF of 450 base pairs encoding 150 amino acids. Using BLAST program, we could find significant similarity between the cloned gene and a trematode myoglobin gene such as Paramphistomum epiclitum, so the protein was named C. sinensis myoglobin(GenBank Accession number AF 461710). Northern blotting was performed to examine the expression of the cloned gene in C. sinensis. When total RNA from C. sinensis was hybridized to the GRCSP2 gene, PRCSP gene, and C. sinensis myoglobin gene, a single band of 1.05 kb, 1.05 kb, and 0.57 kb was detected respectively. The results of Southern blot hybridization indicated that each cloned gene was present within the C. sinensis genome. Purified recombinant GRCSP2 and PRCSP showed high sensitivity for serodiagnosis, 93.3% and 86.7%, respectively. But the cross-reactivity of each recombinant protein was found against paragonimiasis, metagonimiasis, ascariasis, and sparganosis patients' sera. The rate of cross-reactivity using purified recombinant PRCSP was more higher than that using purified recombinant GRSCP2.

Egg‐specific expression of DNMT2 with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis

HYE-JEONG CHO 가천대학교 대학원 2016 국내석사

Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both ofwhichwere elevated in the C. sinensis eggs.However,CsDNMT2and 5 mCweremarginally detected in other histological regions ofC. sinensis adults including ovaries andseminal receptacle. Themethylationsite seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon.Taken together, our data strongly suggest that C. sinensis has preserved the functional DNAmethylation machinery and thatDNMT2 acts as a genuine alternative toDNMT1/DNMT3 to methylateDNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

Gene expression in Clonorchis sinensis and development of DNA vaccine against clonorchiasis

이지숙 Graduate School, Yonsei University 2004 국내박사

간흡충은 담관에 기생하는 흡충류로 우리나라를 비롯하여 대만, 중국 남부, 월남 등에 유행하고 있다. 이 지역에 거주하는 주민 약 700만 명이 간흡충에 감염되어 있는 것으로 보이며 사람들은 피낭유충이 들어 있는 민물고기를 생식하거나 부당하게 조리하여 먹음으로써 간흡충에 감염된다. 간흡충 성충의 유전자 분석은 새로운 약재 개발과 백신 개발에 있어서 유용하다. 간흡충 성충의 cDNA library를 제작하고 무작위로 450개의 클론을 선택, 정제하고 난 뒤 각 염기 서열을 분석한 결과 415개의 클론을 얻을 수 있었다. Basic local alignment search tool (BLAST) programs를 사용하여 National center for biotechnology information (NCBI)에 있는 non-redundant database에서 각 EST의 유사성을 탐색하여 415개의 클론들 중 277개의 클론들이 이미 동정된 유전자에 높은 유사성을 보였고 이 중 220개는 7가지 기능으로 분류할 수 있었다. 나머지 138개는 어떠한 결과에도 일치를 보이지 않았다. 전체 결과를 통해 단백 분해 효소, 지질 결합 단백질, 항원 단백질 외에 진단, 약재, 백신 개발에 유용한 유전자들을 포함하고 있었다. 이러한 EST 분석을 통해 EST가 유전자 발현 분석에 유용한 기술이며 간흡충의 생물학적 특성을 폭넓고 깊이 있게 이해 할 수 있는 다양한 유전자들을 규명할 수 있었다. 이들 다양한 유전자들 중에서 지방산 결합 단백질은 숙주로부터 흡충의 대사에 필요한 긴 사슬 지방산을 세포 내로 전달하는 중요한 역할을 하는 것으로 알려져 있다. 지방산 결합 단백질은 이미 백신 후보 유전자로 알려져 있지만 간흡충에 대한 지방산 결합 단백질에 대한 연구는 없었다. 간흡충의 시스테인 단백 분해 효소는 담도 상피 세포 파괴 및 유충의 이동에 관여하며 병적인 변화를 초래하는 치명적인 요인이다. 간흡충의 지방산 결합 단백질과 시스테인 단백 분해 효소를 DNA 백신을 통해 면역성과 방어 효과에 관한 연구를 실시하였다. 간흡충 지방산 결합단백질과 시스테인 단백분해효소의 cDNA를 발현 벡터 pET28a 에 클로닝 시킨 뒤 대장균에서 과발현 시킨 후, Ni-nitrotriacetic acid (NTA) agarose resin를 사용한 친화크로마토크래피의 방법으로 분리하였다. 간흡충에 감염된 사람의 혈청과 쥐의 혈청에 대해 각각 재조합 지방산 결합 단백질과 시스테인 단백 분해 효소에 대한 항원성을 보였고 이를 통해 간흡충증에 대한 DNA 백신 후보자로의 가능성을 추론할 수 있었다. pcDNA3.1 벡터에 지방산 결합 단백질과 시스테인 단백 분해 효소 cDNA를 클로닝시킨 뒤 대장균에서 과발현 시킨 후 다량의 DNA를 얻은 후 이것을 가지고 각각 간흡충에서는 처음으로 시험관 내와 생체 안에서의 발현을 확인하였다. 지방산 결합 단백질과 시스테인 단백 분해 효소에 대한 DNA 백신을 실시한 쥐에 간흡충의 피낭유충을 감염시킨 뒤 얻은 혈청에 대해 각각 재조합 단백질에 대한 IgG2a 항체를 생성하였고 DNA 백신을 실시한 쥐의 비장을 통해 얻은 비장 세포에서는 IL-4 보다는 IFN-g 의 유의한 증가를 보였다. 이를 통해 Th-2보다는 Th-1에 우세한 면역 반응을 보였다. 또한, 지방산 결합 단백질 DNA 백신을 실시한 쥐에서의 충체 회수율은 40.9% (p<0.05)감소를 보였고 시스테인 단백분해효소에 대한 DNA 백신을 실시한 쥐에서는 31.5% (p<0.05) 감소를 보였다. 이 결과를 통해 간흡충의 지방산 결합 단백질과 시스테인 단백 분해 효소의 각각의 DNA는 실험 동물인 쥐에 피내 접종을 통해 체액성, 세포 매개성 면역을 유도할 수 있었고 차후 간흡충증에 대한 간흡충의 백신 후보자로써의 가능성을 제시하였다. Clonorchis sinensis is a biliary tract parasite, which infects over 30 million people in China, Korea and Southeast Asia through the ingestion of undercooked freshwater fish that harbor the infective metacercariae. The genes expressed in C. sinensis adults were identified in order to develop novel drugs, better diagnostics and vaccines for the parasite. The C. sinensis cDNA library was constructed and DNA sequencing was performed with 450 randomly selected clones. Four hundred fifteen clones contained the amino-acid-encoding sequences. The functions of these genes could be assigned by the DNA sequence homology. The basic local alignment search tool (BLAST) analysis showed that 277 of the 415 clones were strongly matched to previously identified proteins, while the remaining 138 fell into the “no database match” category. Among the clones matching previously identified proteins, 220 putatively identified genes were sorted into 7 functional categories. The dataset included the genes encoding the proteases, a lipid binding protein, the antigen proteins and the other genes of interest from a diagnostics, drug or vaccine development viewpoint. The present express sequence tags (EST) analysis proved to be an effective tool for examining gene expression and identified several important genes for increasing and complementing our knowledge of the biology of C. sinensis.The fatty acid binding protein (FABP) has been known to play an important role in the intracellular transport of long chain fatty acids that are obtained by the fluke from the host. Although FABP has stimulated considerable interest as a vaccine target candidate, the nature of FABP from C. sinensis (CsFABP) remains unclear. Cysteine proteinases of C. sinensis (CsCP) are important virulent factors that induce pathological changes associated with larval migration, and localized biliary epithelial destruction.This study was investigated the immunogenicity and protective efficacy of a DNA vaccine encoding CsFABP or CsCP. The CsFABP or CsCP cDNA was cloned into the expression vector pET28a, expressed in E. coli and the recombinant protein was purified by affinity chromatography. The recombinant CsFABP or CsCP was antigenicity with sera obtained from patients and rats with clonorchisasis. These results suggested that CsFABP or CsCP may be useful as a vaccine candidate for clonorchiasis.Intradermal injection of plasmid DNA carrying the Cs FABP gene (pcDNA-FABP) or the CsCP gene (pcDNA3.1-CsCP) into SD rats elicited both humoral and cellular immune responses. Animals injected with pcDNA-FABP or pcDNA3.1-CsCP developed plasmid-specific antibodies which exhibited a dominance of IgG2a in sera. In addition, the DNA vaccine elicited the production of IFN-g, but not IL-4 in splenocytes, suggesting the induction of a typical Th-1 dominated immune response in rats. The vaccination with CsFABP or CsCP resulted in a significant level of protection in SD rats against challenge infection with C. sinensis metacercariae. These results indicate that pcDNA3.1-CP or pcDNA3.1-CsCP induces cellular and humoral immune response. CsFABP or CsCP may be a good candidate for use in future studies of vaccination against clonorchiasis.