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Galectins from red-lip mullet, Liza haematocheila : Comparative expression profiling and functional aspects as pattern recognition receptor in host immune defense system.

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Abstract
초기면역시스템이 활성화되기 위해서는 다양한 패턴인식수용체들을 통한 선천면역세포들의 병원체 인식이 가장 먼저 이루어진다. 패턴인식수용체가 외부 병원체를 인식하면 세포 내부로의 신호전달이 개시되고 전염증성 또는 항염증성 사이토카인의 분비를 촉진시킨다. galectin 은 당결합단백질로써 세포 내부 또는 외부의 리간드에 결합하거나 외부에서 침입한 병원체 표면의 glycan 에 결합하여 패턴인식수용체로 작용함으로써 선천면역반응을 이끌어내고 그 반응들을 조절하는데 기여하는 것으로 알려져 있다. 이전 연구에서도 galectins 이 호중구, 비만세포, 대식세포와 같은 특정한 선천면역세포의 분화에 있어서 생리학적인 변화를 조절하는데 관련이 있다고 밝혀졌다. 이번 연구에서는 가숭어의 두 가지의 면역관련 유전자인 galectin-1 과 galectin related protein B like 를 규명하였고 조직내 전사체 발현 패턴을 분석하기 위해 면역 자극제인 LPS, poly I:C 그리고 가숭어로부터 분리한 Lactococcus garvieae 를 이용하여 challenge 실험을 진행하였다. 그리고 challenge 후 시간대별로 각 조직을 채취하고 조직으로부터 RNA 를 분리하고 cDNA 를 합성하여 real-time qPCR 을 통해 두 유전자의 각 조직별 mRNA 발현 양상뿐만 아니라 면역 자극 후 시간대별 발현 양상을 확인하였다. 두 유전자의 기능 및 활성을 시험해보기 위해 재조합 단백질을 생산하고 당에 대한 특이성이 있는 지 알아보기 위해 ELISA 방법을 이용하여 sugar binding assay 를 진행하였고, microbial binding assay 와 agglutination assay를 통해 rLhGal-1과 rLhGal B like가 PRR로써 기능을 규명하였다. Galectin-1 과 galectin related protein B like 는 가숭어로부터 유래된 유전자이며 통계에 의하면 가숭어는 한국 어류 전체 소비량 및 생산량의 8 퍼센트를 차지하는 양식 어종이다. 하지만 최근에 한국의 가숭어 양식장에서 Green liver syndrome 이라고 하는 질병이 발병하여 대량 폐사가 잇따라 일어났으며, 원인체는 Lactoccocus garvieae 로 밝혀졌다. 따라서 이번 연구에서는 가숭어 유래 면역유전자에 대한 규명하였고 규명된 유전자가 병원체에 대해 어떠한 작용을 하는 지에 대한 연구가 진행되었으며 이는 앞으로 가숭어의 질병 예방과 가숭어 양식 산업의 발전에 있어서 바탕이 되는 중요한 기초자료가 될 것이다
Activation of immunity depends on the recognition of pathogens by innate immune cells through diverse pattern recognition receptors (PRRs) that initiate their signaling pathway and facilitate secretion of pro- and anti-inflammatory cytokines. Galectins, a family of carbohydrate binding protein have emerged as soluble mediators in infected cells and pattern recognition receptors responsible for evoking and controlling innate immunity. In previous studies, it was demonstrated that various galectin family members are concerned with the physiological turnover of particular innate immune cells; neutrophils, mast cells, macrophages, and dendritic cells. Redlip mullets (Liza haematocheila) are belonged to the Mugilidae family which consists of more than 72 species. The statistics have shown that all species of mullets accounted for 8% of the total consumption and cultivation in Korea. The amount of total fish production of the coastal fishery has been decreased consistently. Recently, high mortality emerged in red lip mullet fishery in Korea, and it was identified that Lactococcus garvieae, which caused green liver syndrome in redlip mullet lead the mass mortality in mullets. Therefore, not only the identification of immune-related genes but study on their response to pathogens is essential for the prevention of diseases and development of the aquaculture industry. In this study, two immune associated genes, galectin-1 and galectin related protein B like were identified from the established redlip mullet cDNA database and defined as LhGalectin-1 and LhGalectin related protein B like. Moreover, mRNA expression patterns of both genes were comparatively analyzed in tissue distribution as well as in several timepoints of specific tissues induced by Lipopolysaccharide (LPS), polyinosinic: polycytidylic acid (poly I:C) and Lactococcus garvieae as immune stimulants. In addition, recombinant LhGal-1 and LhGal B like were examined their carbohydrate binding ability with lactose, galactose and glucose by ELISA method. Furthermore, microbial binding assay and agglutination assay was performed to figure out their functional characterization as a PRRs.
Author(s)
Lim, Chae Hyeon
Issued Date
2020
Awarded Date
2020. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009352
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Lee, Je Hee
Table Of Contents
Summary I
요약문 III
Table of contents V
List of figures VII
List of tables IX
1. Introduction . 1
2. Material and method 3
2.1. Fish rearing, immune challenge, and tissue isolation . 3
2.2. Total RNA extraction and cDNA synthesis 4
2.3. Quantitative real-time PCR . 5
2.4. Identification and in silico analysis of LhGal-1 and LhGal B like sequence. 5
2.5. Cloning and purification of rLhGal-1 and rLhGal B like . 6
2.6. Sugar binding assay 7
2.7. Microbial binding assay 8
2.8. Agglutination assay of LhGal-1 and LhGal B like on bacteria . 9
3. Result and discussion . 9
3.1. Bioinformatic analysis 9
3.2. Tissue-specific distribution of LhGal-1 and LhGal B like expression . 21
3.3. Expression pattern analysis of LhGal-1 and LhGal B like after immune challenge . 24
3.4. Sugar binding ability of rLhGal-1 and rLhGal B like . 28
3.5. Microbial binding activity of rLhGal-1 and rLhGal B like . 32
3.6. Microbial agglutination activity of rLhGal-1 and rLhGal B like 34
4. Conclusion . 36
5. Reference . 37
Degree
Master
Publisher
제주대학교 대학원
Citation
Lim, Chae Hyeon. (2020). Galectins from red-lip mullet, Liza haematocheila : Comparative expression profiling and functional aspects as pattern recognition receptor in host immune defense system.
Appears in Collections:
General Graduate School > Marine Life Sciences
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