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Molecular Insights of two immune related genes via Characterization of TNFα and G-type lysozyme from Seahorse

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Abstract
Lysozyme은 세균의 세포벽에 존재하는 펩티도글리칸의 베타-1,4 결합을 가수분해함으로써 세포벽을 허물어 세균을 죽이는 항균성 효소이다. 동물계에는 닭에서 처음 발견한 chicken-type lysozyme (c-type lysozyme)과 거위에서 발견된 goose-type lysozyme (g-type lysozyme), 그리고 무척추동물에 존재하는 invertebrate-type lysozyme (i-type lysozyme) 등이 존재한다.
Tumor necrosis factor alpha (TNFα)는 cytokines에 속하며, 많은 세포에서 면역조절자로서 다양한 기능을 수행한다. TNFα는 주로 대식세포에서 생성되며, 병원체나 독성의 감염 시inducible nitric oxide synthase (iNOS)나 cyclooxygenase-2 (COX-2) 등의 전염증성 인자를 발현시켜 염증 반응을 일으킴으로써 자극물질로부터 보호하는 중요한 역할을 수행한다. 염증반응을 일으키는 NO와 PGE2는 각각 iNOS와 COX-2에 의해 L-arginin과 arachidonic acid로부터 생성된다.
해마는 실고기과에 속하는 경골어류로서 주로 해안에 서식하고 있다. 그 중에 특히 빅벨리해마는 해마중에서도 가장 크고 아름다운 체색과 체형을 지니고 있어 관상용으로도 가치가 높아 국제해수관상생물 시장에서 인기가 많다. 또한 옛날부터 해마는 약용가치로서 많이 사용되어져 왔으며 이로 인해 일부 국가에서는 남획하고 있다. 뿐만 아니라 지구온난화와 연안의 환경악화로 인해 빅벨리해마가 서식할 수 있는 환경이 축소되어 개체수는 점차 감소하고 있고 호주 및 그 주변국, 중국과 한국에서 인공수정을 통한 양식이 이루어지고 있으나, 그 수가 수요에 비해 현저히 부족한 실정이다. 북해마, 갈귀해마 등에 대해 여러 분야에서 연구되고 있으나, 면역생리학적 연구나 유전자 분석에 대한 연구는 아직 미비하다.
본 연구에서는, 이미 확립되어 있는 cDNA data를 통하여 빅벨리해마(Hippocampus abdominalis)에서 LysG와 TNFα 유전자를 동정하였다. 두 유전자의 아미노산 서열은 ClustalW2 multiple sequence alignment와 EMBOSS needle pairwise sequence alignment를 사용하여 다른 척추동물의 LysG와 TNFα 서열과 비교 분석하였다. 다른 종들과의 orthologous 관계를 확립하기 위해 MEGA 5 software를 사용하여 계통수 분석을 실시하였다. 면역자극에 의해 나타나는 면역반응을 조사하기 위해 LPS, Poly I:C, 그리고 살아있는 병원체인 Edwardsiella tarda과 Streptococcus iniae를 접종하여 각 두 유전자의 시간별 mRNA 발현 양상을 quantitative real-time PCR을 통해 분석하였다. ShLysG와 ShTNFα의 분자적 특성을 조사하기 위해 두 유전자를 각각 pMAL vector에 클로닝한 후 Escherichia coli cells에 형질전환하여 재조합 단백질를 추출 및 정제하였다. rShLysG의 최적활성을 위해 온도별 (10 -60 ℃) 및 pH별 (pH3-12) 활성실험을 수행하였고, turbidimetry method를 이용하여 rShLysG의 최적 활성조건에서 다른박테리아에 대한 항균활성 실험을 실시하였다. rShTNFα의 염증반응에 관여하지 확인하기 위해 쥐의 대식세포인 RAW 264.7 세포에 재조합단백질을 농도별로 처리하여 griess 시약을 통해 NO생성량을 측정하였으며, westen blot 분석을 통해 전염증성 매개인자의 발현 양상을 확인하였다.
ShLysG 유전자는 2개의 catalytic residues, 7개의 N-acetyl-D-glucosamine binding sites, 그리고 catalytic bacterial soluble lytic transglycosylase (SLT) domain이 잘 보존되어 있었으며, 알려진 다른 어류와 높은 상동성과 유사성을 지니고 있었다. pH 4와20에서 가장 높은 활성을 보였고, 항균활성 결과, 3개의 Vibriospp.와 Listeria monocytogenes, 그리고 S.iniae에 대해 높은 항균활성을 보이고 있었다. ShLysG는 모든 조직에서 발현하고 있으며, 특히 신장과 아가미에서 높은 발현을 보이고 있었다. 면역자극을 수행한 결과, 신장과 아가미에서 모든 면역자극에 대해 유의적으로 발현이 증가하는 양상을 보였다.
빅벨리해마의 TNFα 유전자는 transmembrane region과 TNF domain을 가지고 있으며, 다른 어류와 비교한 결과 높은 상동성과 유사성을 가지고 있음을 확인하였다. 또한 모든조직에서 발현하고 있으며 특히 피부와 아가미에서 가장 높게 검출되었다. 면역자극을 수행한 결과, blood과 신장에서 모든 면역자극에 대해 유의적으로 발현이 증가하는 양상을 보였다. 염증조절자로서의 활성을 실험하기 위해 염증유발인자인 NO의 생성량을 측정한 결과, 대식세포에서 농도의존적으로 NO를 생성하였고, western blot 분석결과, iNOS와 COX-2의 단백질발현 역시 농도의존적으로 증가하였다. 이러한 결과로 미루어 볼 때, rShTNFα의 활성은 nuclear factor kB (NB-kB) 경로를 통해 전염증성 매개인자인 iNOS와COX-2를 발현함으로써 NO와PGE2생성을 촉진시켜 염증반응을 일으키는 것이라 사료된다.
종합적으로, 본 연구에서는 빅벨리해마 (Hippocampus abdominalis)으로부터 두 개의 면역관련 유전자, g-type lysozyme (ShLysG)과 Tumor necrosis factor alpha (ShTNFa)를 동정하고 분자적 관점에서 연구하였으며, 더 나아가 어류의 면역학적 연구나 해마의 또 다른 유전자 분석연구에 있어 기초적인 연구자료로 사용될 수 있을 것으로 사료된다.
Lysozyme is a bacteriolytic enzyme protecting the host from bacterial infection which catalyzes the hydrolysis of the β-1-4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan heteropolymers of the bacterial cell wall. Lysozymes are classified into three major distinct lysozyme types in the animal kingdom based on amino acid sequences, biochemical and enzymatic properties: the c-type (chicken or conventional type), the g-type(goose type) and the i-type(invertebrate type) lysozyme.
Tumor necrosis factor alpha (TNFα), also known as cachectin, is a major immunomodulator that is involved in systemic inflammation and make up the acute phase reaction. In response to stimulus such as infection or injury, this cytokine is chiefly secreted by macrophages. Increased levels of excess inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) induced the production of TNFα.
Seahorse (Hippocampus sp.) belongs to the order Syngnathformes and family Syngnathidae resembles the appearance of a horse with a very unique look. In particular, the big-belly seahorse (H. abdominalis) among the seahorses is one of the largest and has ornamental value due to beautiful body color and type. Hence, big-belly seahorse is popular in global ornamental fish market. The overexploitation of seahorse for consumption as well as the exploitation of their habitats decrease the seahorse population and push to the brink of extinction. Limited research has yet been published about molecular characterization and expression profiles of big-belly seahorse's genetic elements.
In this study, TNFα and LysG genes were isolated from a previously established cDNA database of Big-belly seahorse (Hippocampus abdominalis), named as ShTNFα and ShLysG and characterized. The amino acid sequences of ShTNFα and ShLysG were compared with other TNFα and LysG sequences using multiple sequence alignment and EMBOSS needle pairwise sequence alignment, respectively. To investigate the relationship between ShTNFα and ShLysG orthologous with other species, phylogenetic analysis was performed using MEGA5 software. To examine the immune responses upon bacterial and viral stimuli, immune challenge experiments were carried out with LPS, poly I:C, live Edwardsiella tarda and Streptococcus iniae. Subsequently, mRNA expression analysis of ShTNFα and ShLysG genes was performed in a temporal manner. Two genes were cloned into pMAL vector, transformed into Escherichia coli cells and purified in order to use in activity assay of ShTNFα and ShLysG. Antimicrobial activity analysis of rShLysG was carried out using turbidimetry method at pH 4 and 20 ℃. To investigate whether rShTNFα is involved in inflammatory response, NO generation was detected using Griess assay and subsequent western blot analysis from RAW 2647 cells.
ShLysG was consisted with well-conserved catalytic bacterial soluble lytic transglycosylase (SLT) domain (Ala48-Ser168) which contained two catalytic residues (Glu71 and Asp95) and seven of N-acetyl-D-glucosamine binding sites (Glu71, Asp95, Tyr98, His99, Ile117, Tyr145 and Asn146). Identity and similarly of ShLysG was revealed higher relationship to fish LysGs. rShLysG revealed its highest bacteriolytic activity at pH 4 and 20 ℃. Antimicrobial activity of rShLysG was strongly detected towards three Vibrio spp., Listeria monocytogenes and S. iniae. ShLysG was ubiquitously expressed in tissues, most notably in kidney and gill. After the immune challenges, response of ShLysG was significantly increased against all kinds of challenges in kidney and gill.
ShTNFα possessed a transmembrane region (Ile31-Phe23) and a TNF domain (Ala82-Leu243) including six receptor binding sites (Ile100, Asn101, Ser106, Thr158, Ser165 and Asp170) and seven polypeptide binding sites (His84, Phe128, Tyr130, Tyr205, Phe210, Phe237 and Phe241). ShTNFα showed higher degree of identity and similarly with other fish TNFα orthologs. ShTNFα was expressed in all tissues, especially in skin and gill. The mRNA expression of ShTNFα significantly up-regulated upon live bacterial and viral challenges in blood and kidney. NO induction of mouse macrophage RAW 264.7 cells was influenced at its highest concentration (0.25 ㎍/mL) up to the positive control, LPS (10 ng/mL) and increased in a dose-dependent manner. Accordingly, to investigate the downstream events related to inflammatory responses, western blot assay was performed and determined the expression levels of pro-inflammatory factors; iNOS and COX-2. Both expression levels of iNOS and COX-2 were found to increase in a dose-dependent manner. This result implies that ShTNFα may mediate inflammatory responses through NF-B pathway.
In conclusion, in this study, two immune related genes; TNFα and LysG from Big-belly seahorse (Hippocampus abdominalis) were identified and analyzed molecularly and functionally. These genes may play an important role in the immunity of seahorse. Therefore, those genes of seahorse can be used as fundamental research materials in further studies.
Author(s)
고지연
Issued Date
2015
Awarded Date
2016. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007414
Alternative Author(s)
Ko, Jiyeon
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
1. Introduction 1
Chapter I 7
Transcriptional characterization and antimicrobial properties study of G-type lysozyme (ShLysG) from seahorse (Hippocampus abdominalis) 7
2. Materials and Methods 7
2.1. Experimental fish 7
2.2. Big belly seahorse cDNA library construction 7
2.3. In silicoanalysis of ShLysG 7
2.4. Cloning of ShLysG coding sequence 8
2.5. Overexpression and purification of recombinant ShLysG (rShLysG) 9
2.6. Functional characterization of rShLysG 10
2.6.1. Optimal pH and temperature 10
2.6.2. Antimicrobial activity 11
2.7. Immune challenges and tissue collection 11
2.7.1. Fish tissues for the specific distribution analysis 11
2.7.2 Expression pattern of ShLysG after immune challenge 12
2.8. RNA extraction and cDNA synthesis 12
2.9. Quantitative real-time PCR -based ShLysG mRNA expression analysis 12
2.10. Statistical analysis 13
3. Results 14
3.1 Molecular characterization of ShLysG 14
3.2 Phylogenetic analysis of ShLysG 18
3.3 Tissue specific expression of ShLysG mRNA 19
3.4 Expression profile of ShLysG after immune stimulation 20
3.5 Expression and purification of rShLysG 22
3.6 Enzyme activity of rShLysG 23
4. Discussion 26
Chapter II 29
Transcriptional characterization & pro-inflammatory properties study of Tumor necrosis factor α(ShTNFα) from seahorse (Hippocampus abdominalis) 29
5. Material and Methods 29
5.1.Experimental fish 29
5.2. Big belly seahorse cDNA library construction 29
5.3. In silicoanalysis of ShTNFα 29
5.4. Cloning of ShTNFαcoding sequence 30
5.5. Overexpression and purification of recombinant ShTNFα(rShTNFα) 31
5.6. Functional characterization of rShTNFα 32
5.6.1. Cells culture 32
5.6.2. NO production assay 32
5.6.3. Western blot analysis 33
5.7. Immune challenges and tissue collection 33
5.7.1. Fish tissues for the specific distribution analysis 33
5.7.2 Expression pattern of ShTNFαafter immune challenge 34
5.8. RNA extraction and cDNA synthesis 34
5.9. Quantitative real-time PCR-based ShTNFa mRNA expression analysis 35
5.10. Statistical analysis 35
6. Results 36
6.1. Molecular characterization of ShTNFα 36
6.2. Phylogenetic analysis of ShTNFα 39
6.3. Tissue specific expression of ShTNFα mRNA 40
6.4. Expression profile of ShTNFα after immune stimulation 41
6.5. Expression and purification of rShTNFα 43
6.6. Functional characterization of recombinant SHTNFα 44
6.6.1. NO induction assay 44
6.6.2. Western blot 45
7. Discussion 46
References 49
감사의 글 55
Degree
Master
Publisher
제주대학교 대학원
Citation
고지연. (2015). Molecular Insights of two immune related genes via Characterization of TNFα and G-type lysozyme from Seahorse
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