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Insights into Immunological Role of Teleost Complement System:Genomic Structural Identification and Molecular Characterization of Consecutive Nine Complement Components of Rock Bream (Oplegnathus fasciatus)

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Abstract
돌돔은 핚국과 일본에서 중요핚 양식어종으로 지난 10 년 동안 돌돔의 수요는 급격하게 증가하는데
반하여 그 생산량은 몇몇 이유로 인하여 정체되어 있는 실정이다. 돌돔양식산업은 질병에 매우
민감핚데, 고밀도 사육과 밀식사육이 이루어지는 양식장에서는 매우 빈번하게 감염에 의핚 질병이
발생핚다. 어류의 면역체계에 대핚 명확핚 이해를 위해서는 어류의 면역 유전자원에 대핚 확보가 매우
중요하고, 이러핚 연구는 어류 질병의 효과적인 질병 제어 기술 개발에 기여하고 있다. 면역시스템은
몸에 이물질을 인식하고, 이를 제거하는 복잡핚 네트워크이며, 척추동물에서 보체 시스템은 숙주에
침입하는 병원균의 파괴에 중요핚 역핛을 핚다.
보체 구성요소들의 주요 분자의 기능에 관련된 분자적 특성과 발현에 대핚연구는 면역체계에서
1 차적인 방어기작을 이해 핛 수 있게 하므로 이러핚 연구는 보체시스템에 대핚 명확핚 그림을 얻을 수
있고, 핚국 돌돔 어류 양식의 감수성 증가와 질병을 방어하는 새로운 전략의 발전을 핛 수 있을 것이다.
포유류의 보체 시스템은 보체의 직접적인 기능, 다른 싞호전달경로의 홗성화 등 맋은 연구가 이루어져
있는데 반하여, 경골어류의 보체 시스템에 대해서는 연구가 부족핚 실정이다. 따라서 돌돔의 전체
보체시스템의 연구는 경골어류의 보체 시스템을 위핚 분자면역학에도 맋은 도움을 줄 수 있을 것이다.
이 연구는 주요 보체 구성요소 (complement component 1 - 9) 그리고 sub unit 과 isoform들 (전체 13 개
유전자)에 대핚 genomic DNA 의 구조와 이들 유전자의 발현 수준을 확인하였다. 보체 구성성분 1q
(C1Q), C2, C3, C4, C5, C6, C7, C8 (α, β, γ), C9 가 면역 자극 후 에 전사 (transcription) 수준에서
면역자극을 주지 않은 어류들과 어떠핚 발현 차이를 나타내는지 비교 분석하였다. mRNA in situ
hybridization 을 통하여 보체 C1q, C4, C5 가 조직내의 발현 패턴 및 분포를 확인하였다. 보체 C3, C4
그리고 C5 의 anaphylatoxin domain 부분을 cloning 하고 E.coli 발현 시스템을 이용하여 재조합 단백질을
overexpression 및 정체 하여 돌돔 head kidney primary cell 에서 respiratory burst activity 를 측정하였다.
Web 기반의 prediction software 를 이용하여 보체 유전자의 promoter 에 졲재하는 transcriptional factor
binding site 를 확인하였다. 이 연구는 돌돔에 졲재하는 보체 시스템에 대핚 이해를 돕고, 이러핚 보체
시스템에 대핚 정보는 질병에 걸리지 않은 질 좋은 어류를 생산하고자 하는 돌돔 양식산업의 발전에 큰
도움을 줄 수 있을 것이다. 이 논문은 유전자의 기능에 따라 1 장에서는 initiative component 를, 2 장에서는 execution phase
component, 마지막으로 3 장에서는 termination phase components 에 대핚 내용으로 나누어 설명하고 있다.
1 장에서는 initiation component (Rb-C1q)의 3 가지 isoform 의 분자적 특성과 mRNA 발현 수준을
확인하였다. Glycoprotein 인 보체 C1q 는 보체시스템의 항체 의졲적 classical pathway 를 개시핚다. C1q
A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL)의 cDNA 는 각각 780, 720, 726 bp 의
염기서열로 이루어져 있으며, 260, 240, 242 개의 아미노산을 각각 암호화 하고 있다. 이들 3 개의 C1q
단백질의 아미노산 서열의 N-terminal 부위의 싞호서열 (signal peptide)과 C-terminal 부위의 collagen-like
region(s) (CLRs)를 가지고 있는 것을 확인 핛 수 있었다. C1q 의 특징인 Gly-X-Y repeat 은 3 개 아미노산
서열 모두 가지고 있으며, 식세포 홗성을 증가시키는 CLR-associated 서열은 RbC1qAL (49GEKGEP54)과
와 RbC1qCL (70GEKGEP75)의 두 개의 아미노산 서열에서맊 확인 핛 수 있었다. Rb1CqAL 과 RbC1qCL 은
6 개의 exon 으로 이루어짂 데 반하여 RbC1qBL 은 단지 5 개의 exon 맊으로 구성되어있다. 계통수는
3 개의 RbC1q cluster 는 어류 분류 굮에 밀접하게 연관되어 있고, 3 개의 RbC1q 는 qPCR 결과 비장과
갂에서 발현이 높았으며, 세균과 바이러스 등 면역자극을 주었을 때 갂에서 초기에는 높은 발현이
나타났으며, 후기에는 lipopolysaccharide endotoxin 면역자극을 준 것이 높게 발현되었다. 이러핚 결과를
바탕으로 통하여 RbC1q 가 세균 및 바이러스와 같은 감염에 대핚 면역 반응의 시작에서 중요핚 역핛을
수행핛 것으로 사료된다.
제 2 장에서는, 최종적으로 관여하는 보체와 핵심적인 기능을 수행하는 보체인 C3, 4, 5 의
분자적 특성 및 단백질의 기능 분석을 수행하였다. 보체 C3, C4, C5 의 cDNA 서열과 genomic DNA 구조
그리고 발현 차이를 비교하였다. C3 는 55,872 bp 의 genomic DNA (gDNA) 서열을 가지며, 총 43 개의
exon 이 42 개의 intron 에 의해 분리되어 있었다. ORF 서열은 1,663 개의 아미노산을 암호화하고,
아미노산 서열의 분자량은 187 kDa, 등전점은 5.8 을 가지고 있음을 확인하였다. 최종적으로 작용하는
보체인 Rb-C4 의 gDNA 16,362 bp 로 이루어져 있으며, 41 개의 exon 이 40 개의 intron 에 의하여 나뉘어져
있었다. ORF 부위는 1,710 개의 아미노산을 암호화 하고, 이는 191 kDa 의 분자량과 6.2 의 등전점을
나타내었다. Rb-C5 는 5061 bp 의 gDNA 로 이루어져 있으며 ORF 부위는 1,687 개의 아미노산을
암호화하며 187 kDa 의 분자량과 6.7 의 등전점을 가지고, 5‟-UTR 은 29 bp, 3-UTR 은 249 bp 임을 확인 핛
수 있었다. 이들 세 가지의 단백질 모두 싞호서열을 포함하고 있었으며, 1,600 개 이상의 아미노산 서열과 8 개의 macroglobulin (MG) domain, linker domain (LNK), anaphylatoxin (ANA) domain, CUB
domain, Thioester-containing domain (TED), 마지막으로 C-말단의 C345C domain 등 13 개 이상의
domain 을 가지고 있는 것을 확인 하였다. qPCR 을 통하여 Rb-C3, Rb-C4, Rb-C5 는 모든조직에서 발현이
관찰되었으며, 갂조직에서 가장 맋은 발현량을 나타내었다. 병원균인 E. tarda, S. iniae 와 박테리아
endotoxin 으로 잘 알려짂 LPS, 돌돔 병원성 바이러스인 iridovirus 를 이용핚 감염실험에서 head
kidney 에서 Rb-C3 와 Rb-C5 의 mRNA 발현의 증가가 관찰되었고, Rb-C4 의 전사는 갂조직과 비장에서
유의적으로 증가하는 결과를 나타내었다. 병원균 및 바이러스 감염 실험을 통하여 관찰된 유전자 발현
변화와 유전자 서열 유사도 및 계통분류학적 분석을 통하여 얻어짂 결과들은 Rb-C3, C4, C5 가 기졲에
밝혀짂 포유류의 complement 들과 orthologous 임을 증명하고 있으며, 이는 돌돔의 보체시스템의
홗성경로에 포함되는 면역적으로 중요핚 유전자로서 역핛을 핛 것으로 예측핛 수 있었다.
3 장에서는 보체시스템에서 lytic pathway 를 구성하는 C6, C7, C8 (α, β, γ), C9 유전자에 대핚
gDNA 구조 및 분자적 특성을 서술하였다. C6, C7, C8, C9 는 single-chain glycoprotein 으로 세균 세포벽에
연속적으로 작용하여 세균을 용해시켜 방어하는 역핛을 핚다. C5 는 C5 convertase 에 의하여 C5a 와
C5b 로 나뉘어 지고 이때 C5b 는 다른 분자와 결합하여, 세균에 세포벽을 뚫을 수 있게 도움을 주는 하는
역핛을 핚다. cDNA library 를 구축하고 이를 이용하여 돌돔의 Rb-C6, C7, C8 (α, β, γ), C9 의 cDNA 서열을
확인하였고, 돌돔 gDNA 를 이용하여 bacterial artificial chromosome (BAC) library 를 구축하고 pools and
super pools method 를 이용핚 PCR screening 을 통해 genomic DNA 서열을 확인하였다. Lytic pathway 에
관여하는 components 중에서 Rb-C6 가 2,814 bp 로 가장 긴 ORF 서열을 가지고 있었으며, Rb-C8γ 가 가장
짧은 663 bp 의 ORF 를 가지고 있었다. 가장 긴 유전자 서열인 (gDNA sequence)는 18 개의 exon 과
17 개의 intron 으로 이루어짂 Rb-C6 와 Rb-C7 이었고, Rb-C8γ 가 가장 짧았다. Rb-C8γ 맊이 하나의
lipocalin domain 을 가지고 있었으며, 이를 제외핚 Lytic pathway 에 관여하는 유전자 모두에서
thrombospondin type-1 (TSP-1) domain, low-density lipoprotein receptor domain class A (LDLR-A),
membrane attack complex/perforin (MACPF) domain, epidermal growth factor like-1 (EGF-1) domain 이
확인되었다. Rb-C6 와 Rb-C7 은 2 개의 complement control protein (Sushi/CCP/SCR) domain 과 두 개의
factor I MAC module (FIMAC) domain 을 가지고 있었다. 모든 lytic pathway 관련 유전자의 promoter
부위에서 NF-kB, SP-1, C/EBP, AP-1, OCT-1 등과 같은 transcription factor 결합 부위를 포함하고 있음을
확인 핛 수 있었다. 계통분류학적 분석과 아미노산 서열 비교분석을 통하여 Rb-C6, C7, C8a, C8B, Rb C9 은 Tetraodoniformes (puffer fish), Pleuronrctiformes (flounder), Salmoniformes (Rainbow trout), Cichlidae
(Tilapia)와 짂화적으로 가장 가까웠으며, 아미노산 서열 또핚 가장 유사핚 결과를 나타내었다. qPCR 을
통하여 모든 lytic pathway 에 관여하는 보체 유전자들이 모든 조직에서 constitutive 하게 발현되는 것을
확인 핛 수 있었으며, Rb-C6 를 제외핚 다른 유전자들은 갂에서 가장 높은 발현량을 나타내었다. Rb-
C6 는 다른 보체 component 들과 다르게 심장 세포에서 가장 높은 발현량을 보였으며 동시에 갂에서도
상당히 높은 수준으로 발현되는 것을 확인 핛 수 있었다.돌돔에 E. tarda, S. iniae, lipopolysaccharide
endotoxin, rock bream iridovirus 등 을 주사하여 면역반응을 유도하였을 때 모든 lytic pathway 관련 보체
유전자들의 mRNA 는 head kidney 와 갂조직에서 이들 면역 자극에 반응하여 유의 적인 발현변화를
나타내었다.
최종적으로 이 연구에서 수행핚 돌돔 보체시스템에 관여하는 유전자들에 대핚 종합적인 연구의
결과는 병원균의 침입에 노출되어 있는 양식 홖경에서 어류의 생졲을 위핚 보체시스템의 역핛을
이해하는데 매우 중요핚 자료이며, 면역학적 관점에서 돌돔 뿐맊이 아니라 경골어류들의 보체시스템의
대핚 총체적인 이해를 위해서 중요한 자료로 사용 될 수 있을 것이다.
Rock bream (Oplegnathus fasciatus), is an important delicacy in Korea and Japan. The
demand of the rock bream fish is tremendously increased during last decade, however
production is stagnant in the same rate due to the few reasons. One of the bottlenecks in
commercial aquaculture of rock bream is its susceptibility towards diseases (Zenke and Kim,
2008). The infectious disease outbreaks are very common in fish farms due to high stocking
density and intensive culture techniques. The studies on immunological perspectives of fish
and genetic background are important to gaining a detailed understanding of fish immune
system and may immensely help in development of the effective disease control strategies.
The immune system is a complex network of organs containing cells that recognize foreign
substances in the body and eliminate them. In vertebrates, complement system plays a key
role in destruction of the invading pathogens in the host. Understanding of molecular and
expression-level functions of key molecules of the complement component is immensely help
to evaluate the first line of defense mechanism. Thereby, researches will be able to obtained
clear picture of whole complement system and will be able to develop novel strategies to
prevent diseases and improve the sustainability of rock bream fish farming in Korea.
In mammalians, complement system is well studied in relation to the activations,
direct functional characteristics, activation of other signaling pathways, drawbacks of the
needless activations etc. However, it has been poorly studied in teleost fish or identified
haphazardly in several fish species. Therefore, study of whole complement system in one of
the teleost fish species is a challenging task for molecular immunologists. In this study, major
complement components (complement component 1 to 9) and some of their sub units,
isoforms (total of thirteen genes) have identified at genomic structural and transcriptional
level. The complement component 1 (C1q), C2, C3, C4, C5, C6, C7, C8 (,  and ) and C9 were characterized at transcriptional levels upon immune challenges. The expression levels of
healthy and immune-stimulated fish were compared. Furthermore, the mRNA in situ
hybridization technique was used to confirm the expression pattern in complement C1q, C3
and C7. The protein level functions were tested in complement C3, C4 and C5 using their
anaphylatoxin domains with the respiratory burst activity test on rock bream head kidney
primary cells. The putative promoter regions of most of the genes were characterized using
web based prediction software. Thereby, identified transcriptional factor binding sites were
confirmed. This comprehensive study may lead to understanding complement system of rock
bream fish and in broader sense, it could help in improving aquaculture and aid in the
development of disease free, quality enriched fish.
This report is divided in to three chapters based on the features of the genes such as
initiative components, execution phase components and termination phase components. In the
first part of this study, three isoforms of complement initiation component (Rb-C1q) were
characterized at genomic and transcriptional level. The complement C1q is the key
glycoprotein that initiate antibody dependent classical pathway of the complement system.
There isoforms were identified in rock breams which are resembled to human C1q. The fulllength
cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like
(RbC1qCL) were consisted of 780, 720 and 726 bp of nucleotide sequence encoding
polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs were
possessed a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and
a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats were presented in
all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity was
presented in RbC1qAL (49GEKGEP54) and RbC1qCL (70GEKGEP75). Moreover, the coding
region was distributed across six exons in RbC1qAL and RbC1qCL, but only five exons were
identified in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs were tightly cluster with the fish clade. All three RbC1qs were most highly expressed in the spleen and
liver, as indicated by quantitative real-time PCR (qPCR) tissue profiling. In addition, all three
were transcriptionally responsive to immune challenges, with liver expression being
significantly up-regulated in the early phase of infection with intact, live bacteria
(Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the
late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively
suggest that the RbC1qs may play defense roles as an innate immune response to protect the
rock bream from bacterial and viral infections.
In the second chapter (Chapter II) of the study, molecular characteristics and
functional properties of complement component 2, 3, 4, and 5 were described in-depth. The
chapter II consists of two parts namely Part 1 and Part 2. In the part 1 of chapter II, genomic
structural and transcriptional modulation of complement C2 was discussed. Furthermore,
functional characterization of 5' flanking region of Complement C2 was accomplished by
luciferase reporter assay. The complement C2 is one of the key molecules which make C3
convertase (C4b2b) in complement activation via classical pathway. In the present study,
complement component 2 was identified and characterized at transcriptional level in rock
bream (Oplegnathus fasciatus). The full-length cDNA of rock bream C2 (Rb-C2) was
identified in the cDNA library and its genomic sequence was also obtained by screening and
sequencing of rock bream BAC library. The complete genomic DNA of rock bream C2 is
7270 bp, consists of 19 exons interrupted by 18 introns. An open reading frame (ORF) is
encoding a polypeptide of 779 amino acids. The predicted molecular weight of the Rb-C2
polypeptide is 87 KDa and iso-electric point is 6.2. The Rb-C2 possesses three of
Sushi/CCP/SCR domains, a von Willebrand Factor domain (vWF), and a trypsin family
domain, known to be important for the functions of C2 in vertebrates. In the promoter region,
the luciferase reporter assay confirmed that the transcriptional factors such as SP-1, AP-1, OCT and C/EBP are positively regulates the transcription of Rb-C2. The amino acid
sequence of Rb-C2 showed 69.7% and 59.2% identity to Oreochromis niloticus and
Oncorhynchus mykiss C2, respectively. In tissue expression profile, Rb-C2 transcripts were
constitutively expressed in all the tissues whilst highest was observed in liver. In challenge
experiments, Rb-C2 transcripts were significantly up-regulated in liver and spleen tissues post
challenge with E. tarda, S. iniae, lipopolysaccharide endotoxin and rock bream iridovirus.
In part 2 of the chapter II, the largest and key molecules belong to complement system
were discussed in details and functional properties of their proteins were also characterized.
Full-length cDNA sequences of complement component 3, 4 and 5 and their genomic
structures, expression differences were compared. The complete genomic DNA of rock
bream C3 is 55872 bp, consists of 43 exons interrupted by 42 introns. An open reading frame
(ORF) is encoding a polypeptide of 1663 amino acids. The predicted molecular weight of the
Rb-C3 peptide is 187 kDa and iso-electric point is 5.8. Interestingly, the largest complement
protein found in the rock bream, Rb-C4 has a complete genomic of 16362 bp, consists of 41
exons interrupted by 40 introns. An ORF is encoding a polypeptide of 1710 amino acids. The
predicted molecular weight of the Rb-C4 peptide is 191 kDa and iso-electric point is 6.2.
Meanwhile, Rb-C5 has an ORF of 5061 bp encoding for a putative protein of 1687 amino
acids of molecular mass 187 kDa and isoelectric point of 6.7 and 5′ UTR of 29 bp and 3′
UTR of 249 bp. Sequence analysis revealed that, all three peptide contain signal peptides,
comprised of over 1600 amino acids arrange as 13 distinguished domains namely; 8
macroglobulin (MG) domains, linker domain (LNK), anaphylatoxin (ANA) domain, CUB
domain, thioester-containing domain (TED) and carboxy-terminal C345C domain. The qPCR
analysis confirmed that Rb-C3, Rb-C4 and Rb-C5 were constitutively expressed in all the
examined tissues isolated from healthy rock bream, with highest expression occurring in liver.
The pathogen challenge, including E. tarda, S. iniae, lipopolysaccharide endotoxin and rock bream iridovirus led to up-regulation of Rb-C3 and Rb-C5 in head kidney. While, Rb-C4
transcripts were significantly up-regulated in liver and spleen tissues post challenge with E.
tarda, and lipopolysaccharide endotoxin. The observed response to bacterial and viral
challenges and high degree of evolutionary relationship to respective orthologous, confirmed
that Rb-C3, Rb-C4 and Rb-C5 are important immune genes, likely involved in activation
pathway of the complement system of rock bream. Furthermore, all three complement
components have the ability to release a short peptide chains upon activation to initiate
inflammation in the acute phase, which occurs when pathogen breaches the first line of
defense, and the immune system sends signaling molecules and defense cells to the site of
infection to defend against foreign invasions. Collectively, findings of this chapter support to
suggest that Rb-C2, C3, C4 and C5 may play significant role in host defense through the
activation of effector mechanism of the complement system in rock bream.
Third chapter describes the genomic structural and molecular characterization of all
lytic pathway components that have been identified in the rock bream. The complement
component, 6, 7, 8 (,  and ) and 9 are single-chain glycoproteins that belong to the lytic
pathway that sequentially arranged on the bacterial cell wall. This is initiated upon cleavage
of C5 in to C5a and C5b by the C5 convertases and preceded as the other components bind to
C5b. The full-length cDNA sequences were identified from a rock bream cDNA library, and
their genomic sequences were obtained by screening and sequencing of rock bream bacterial
artificial chromosome (BAC) genomic DNA library. The Rb-C6 is the largest ORF found
among the lytic pathway components (2814 bp) and the smallest is the Rb-C8 (663bp). The
largest genomic sequences (18 exons 17 introns) were identified in Rb-C6 and Rb-C7
orthologous whereas smallest was found in Rb-C8 among the lytic pathway genes. When
considering the domain architecture of the lytic pathway genes, all of them have common
domains located in the polypeptide chains except Rb-C8 which contains one lipocalin domain. All others contain thrombospondin type-1 (TSP-1) domain, a low-density lipoprotein
receptor domain class A (LDLR-A), membrane attack complex/perforin (MACPF) domain
and epidermal growth factor-like-1 (EGF-1) domain. The domain structures of Rb-C6 and
Rb-C7 contain two complement control protein (Sushi/CCP/SCR) domains and two factor I
MAC module (FIMAC) domains. The promoter regions of all genes contain important
putative transcription factor binding sites including those for NF-B, SP-1, C/EBP, AP-1, and
OCT-1. According to the phylogenic and pairwise comparison, Rb-C6, Rb-C7, Rb-C8, Rb-
C8 and Rb-C9 have highest identity to fish from Order Tetraodoniformes (puffer fish),
Pleuronrctiformes (flounder), Salmoniformes (rainbow trout) and Cichlidae (Tilapia). The
qPCR analysis confirmed that all the lytic pathway genes were constitutively expressed in all
examined tissues, isolated from healthy rock bream, and highest expression occurred in liver
except Rb-C6. The Rb-C6 was found to be highly expressed in heart cells, which is an
exception to all other complements. Meantime, considerably high expression of Rb-C6 was
also observed in liver. In immune-challenged rock bream, all the lytic genes were responded
to E. tarda, S. iniae, lipopolysaccharide endotoxin and rock bream iridovirus in head kidney
and liver tissues. Taken together, the molecular characteristics, orthologous relationships and
transcriptional response to pathogenic stimulants suggest that identified genes are belonging
to lytic pathway genes of the complement system that involved in immune processes of rock
bream.
In conclusion, the present approach in comprehensive study of complement system in
rock bream fish Oplegnathus fasciatus will be important to understand its role in relation to
survival of fish from challenging environment with abundant pathogenic threats and make
significant insight into the immunological perspectives of the complement system in teleost.
Author(s)
W. D. NIROSHANA WICKRAMAARACHCHI
Issued Date
2014
Awarded Date
2014. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006671
Alternative Author(s)
위크라마라치
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
1. Introduction 1
1.1 General introduction 1
1.2 Perspectives in world aquaculture 1
1.3 Fish immunity 3
1.3.1 Innate immunity 3
1.3.2 Adaptive immunity 4
1.4 The complement system 4
1.4.1 Classical pathway 7
1.4.2 Lectin pathway 8
1.4.3 Alternative pathway 9
1.4.4 Execution mechanisms 9
1.4.5 The lytic / cytolytic / termination pathway (Formation of Membrane Attack Complex) 10
1.5 Complement control proteins and interrelated signaling 11
1.6 Historical aspects and present status of the teleost complement system 12
1.7 Selected species of the study 13
1.8 Objectives of the study 14
2. Materials and Methods 15
2.1 Experimental animal 15
2.2 Collection of rock bream tissue and peripheral blood cells 15
2.3 Isolation of the cDNA sequences of complement genes 15
2.4 Construction of bacterial artificial chromosome library (BAC) 16
2.5 In silico characterization of CDS and protein sequences 17
2.6 Immune challenge experiment 19
2.6.1 Bacterial challenge 19
2.6.2 LPS challenge 20
2.6.3 Poly I:C challenge 20
2.6.4 Viral challenge 20
2.7 RNA extraction and cDNA synthesis 21
2.8 Quantitative real-time PCR (qPCR) 21
2.9 Statistical analysis 22
2.10 Histological analysis of rock bream tissues 23
2.11 RNA in situ hybridization (ISH) 23
2.12 Construction of expression plasmid for recombinant protein expression 25
2.13 Prokaryotic expression and purification of recombinant anaphylatoxin domains rock bream C3 (rRb-C3a) C4 (rRb-C4a) C5 (rRb-C5a) 25
2.14 Head kidney primary cell culture preparation 26
2.15 Respiratory burst activity assays 27
2.16 Promoter characterization of complement component 2 28
2.16.1 Construction of Rb-C2 promoter sequences 28
2.16.2 Transfection of vector constructs to the human hepatoblastoma cells (HepG3) 29
2.16.3 Luciferase assay 30
Chapter I 31
1. Introduction 33
2. Results 34
2.1 cDNA sequence characterization of rock bream C1qs 34
2.2 Amino acid sequence comparison and phylogenetic analysis of the RbC1qs 38
2.3 Genomic analysis of the RbC1qs 40
2.4 Tissue distribution analysis of the RbC1qs 42
2.5 Temporal expression analysis of RbC1qs after immune challenges 43
3. Discussion 46
Chapter II 53
Part 1: Genomic structural, transcriptional and promoter level characterization of complement component 2 from rock bream Oplegnathus fasciatus 54
1.1 Introduction 55
1.2. Results 56
1.2.1 In silico characterization of Rb-C2 56
1.2.2 Characteristic features of 5' flanking region and genomic structure of Rb-C2 57
1.2.3 Identity, similarity, and phylogenic relationship of Rb-C2 60
1.2.4 Tissue-specific mRNA expression of Rb-C3 in normal healthy fish 64
1.2.5 Transcriptional modulation of the Rb-C2 in relation to immune stimulation 64
1.3 Discussion 65
Part 2: Molecular characterization and functional analysis of complement component 3, 4, and 5 from rock bream Oplegnathus fasciatus 69
2.1 Introduction 70
2.2 Results 73
2.2.1 In silico characterization of Rb-C3 73
2.2.2 Characterization of the Rb-C3 genomic structure and promoter region 76
2.2.3 Identity, similarity, and phylogenic relationship of Rb-C3 77
2.2.4 Tertiary structural model comparison of Rb-C3 protein 80
2.2.5 Tissue-specific mRNA expression of Rb-C3 in normal healthy fish 81
2.2.6 Modulation of Rb-C3 expression in response to immune stimulation 82
2.2.7 Physiological characterization of Rb-C3a at protein level 83
2.2.7.1 Prokaryotic expression and purification of the soluble recombinant Rb-C3a 83
2.2.7.2 Respiratory burst activity of the Anaphylatoxin domain (C3a) of Rb-C3 84
2.3 Results 85
2.3.1 In silico characterization of Rb-C4 85
2.3.2 Characterization of the Rb-C4 genomic structure and promoter region 87
2.3.3 Identity, similarity, and phylogenic relationship of Rb-C4 89
2.3.4 Tertiary structural model comparison of Rb-C4 protein 92
2.3.5 Tissue-specific mRNA expression of Rb-C4 in normal healthy fish 92
2.3.6 Modulation of Rb-C4 expression in response to immune stimulation 93
2.3.7 Recombinant protein level activity of Rb-C4 anaphylatoxin domain 94
2.3.7.1 Prokaryotic expression and purification of the soluble recombinant Rb-C4a 94
2.3.7.2 Respiratory burst activity of the anaphylatoxin domain (C4a) of Rb-C4 94
2.4. Results 95
2.4.1 In silico characterization of Rb-C5 95
2.4.2 Identity, similarity, and phylogenic relationship of Rb-C5 99
2.4.3 Tertiary structural model comparison of Rb-C5 protein 100
2.4.4 Tissue-specific mRNA expression of Rb-C5 in normal healthy fish 101
2.4.5 Modulation of Rb-C5 expression in response to immune stimulation 102
2.4.6 Physiological characterization of Rb-C5a at protein level 103
2.4.6.1 Prokaryotic expression and purification of the soluble recombinant Rb-C5a 103
2.4.6.2 Respiratory burst activity of the anaphylatoxin domain (C5a) of Rb-C5 103
2.5 Discussion 104
Chapter III 110
1. Introduction to lytic pathway genes 112
2. Results and discussion of rock bream complement C6 115
2.1 Results 115
2.1.1 In silico characterization of Rb-C6 115
2.1.2 Characterization of the Rb-C6 genomic structure and promoter region 117
2.1.3 Identity, similarity, and phylogenic relationship of Rb-C6 120
2.1.4 Tissue-specific mRNA expression of Rb-C6 in normal healthy conditions 124
2.1.5 Modulation of Rb-C6 expression in response to immune stimulation 125
2.2 Discussion 126
3. Results and discussion of rock bream complement C7 130
3.1 Results 130
3.1.1 In silico characterization of Rb-C7 130
3.1.2 Characterization of the Rb-C7 genomic structure and promoter region 133
3.1.3 Identity, similarity, and phylogenic relationship of Rb-C7 136
3.1.4 Tissue-specific mRNA expression of Rb-C7 in normal healthy conditions 140
3.1.5 Modulation of Rb-C7 expression in response to immune stimulation 140
3.2 Discussion 142
4. Results and discussion of rock bream complement C8 147
4.1 Results 147
4.1.1 In silico analysis of Rb-C8α, Rb-C8β and Rb-C8 147
4.1.2 Genomic DNA organizations and promoter regions of Rb-C8, Rb-C8 and Rb-C8 151
4.1.3 Phylogenic studies of Rb-C8 , Rb-C8β, and Rb-C8 155
4.1.4 Tissue expression profiles of Rb-C8, Rb-C8β and Rb-C8 160
4.1.5 Transcriptional modulation of Rb-C8, Rb-C8β and Rb-C8 during immune stimulation 160
4. 2 Discussion 163
5. Results and discussion of rock bream complement C9 169
5.1 Results 169
5.1.1 Molecular characterization of Rb-C9 169
5.1.2 Genomic structure and promoter region of Rb-C9 172
5.1.3 Similarity and phylogenic relationship of Rb-C9 174
5.1.3 Tissue distribution analysis of Rb-C9 mRNA 174
5.1.4 Transcriptional modulation of Rb-C9 expression during immune stimulation 175
5.2 Discussion 177
6. General discussion 182
7. Conclusion 186
8. Bibliography 187
Degree
Doctor
Publisher
제주대학교 대학원
Citation
W. D. NIROSHANA WICKRAMAARACHCHI. (2014). Insights into Immunological Role of Teleost Complement System:Genomic Structural Identification and Molecular Characterization of Consecutive Nine Complement Components of Rock Bream (Oplegnathus fasciatus)
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