돌돔과 참돔 면역유전자의 특징과 발현

Abstract

참돔과 돌돔에서 iridovirus 감염 시 pro-inflammation gene인 IL-1β와 IL-1β에 의해 유도되는 chemokine인 IL-8 그리고 COX-2 gene과 interferon에 의해 유도되는 Mx gene의 발현량의 차이를 qRT-PCR의 상대정량을 이용하여 비교하여 보았으며, 아직 염기서열이 분석되지 않은 참돔의 Mx, IL-8 그리고 COX-2 gene과 돌돔의 IL-8 gene의 full sequence를 분석하였다. rsbMx1, rsbMx2, rsbIL-8 rbIL-8 그리고 rsbCOX-2 gene의 cDNA의 전체 염기서열은 각각 2381 bp, 2806 bp, 865 bp, 888bp 그리고 2528bp 이며, rsbMx1과 rsbMx2 gene은 114bp의 5‘ UTR, 1881 bp의 ORF 그리고 126 bp의 3‘ UTR, 1914bp의 ORF 그리고 766 bp의 UTR로 구성 되었어 있으며, 12개의 exon과 11개의 intron으로 구성되어있었다. rsbIL-8 과 rbIL-8 gene은 188 bp의 5’ UTR, 298 bp의 ORF 그리고 379 bp의 3‘ UTR과 203 bp의 5’ UTR, 312 bp ORF 그리고 373 bp의 3‘ UTR로 구성되어 있으며, 4개의 exon과 3개의 intron으로 구성되어있었다. rsbCOX-2 gene은 92 bp의 5’ UTR, 1827 bp의 ORF 그리고 609 bp의 3‘ UTR로 구성되어있으며, 10개의 exon과 9개의 intron으로 구성되어있었다. 또한 iridovirus 감염에 따른 민감어와 둔감어의 immune gene의 발현을 알아보기위해 참돔과 돌돔에 iridovirus와 immuno stimulant인 poly I:C를 challenge하고 1일, 3일, 7일째 immune gene의 발현을 비교하여 보았다. 그 결과 참돔과 돌돔에서 poly I:C를 주사하였을 경우 1일째 rsbMx1, rsbMx2 그리고 rockbream Mx gene이 각각 약 21.5, 19.8 그리고 19배 증가하여, 두 어종모두 비슷하게 증가하였다. 반면 iridovirus를 challenge 하였을 때는 3일째 rsbMx1와 rsbMx2 gene은 최고치를 보였으며 각각 약 88, 208배 증가하였으며, rockbream Mx gene은 7일째 약 33배증가한 것을 확인 할 수 있었다. 하지만 IL-8, COX-2 그리고 IL-1β gene의 발현은 확인 할 수 없었다. 이러한 어류 면역유전자 분석과 iridovirus 감염에 대한 면역유전자 발현 분석은 향후 iridovirus의 치료법이나 예방법을 연구하는데 기초적인 연구 자료로 사용될 것으로 사료된다.

The type I interferon (IFN) system is one of the most important mechanisms for antiviral defence. In the type I IFN response, many IFN-related proteins are induced for establishing an antiviral state. As a one of the IFN-related protein, Mx protein have been shown to inhibit virus replication. While interleukin-1 is a important pro-inflammatory cytokines often used as markers of an activated inflammatory response. One of the genes regulated by IL-1 is IL-8, which is a member of the CXC chemokine family. Also increased IL-1 has been attributed to the induction of novel Cyclooxygenase (COX) isoform (COX-2), distinct from the well characterized constitutive activity (COX-1) and playing an important role in immune regulation. COX-2 enzyme is induced by various inflammatory signals, including cytokines, growth factors, and lipopolysaccharide. Iridovirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogenesis, little is known interaction between iridovirus and the fish immune system. Therefore, three important immune genes, Mx, IL-8 and COX-2 genes were cloned and sequenced in red sea bream and rock bream. Using degenerated primers designed from known sequences, a cDNA fragment was amplified by PCR and elongated by 5'- and 3'- RACE-PCR to give the full-length coding sequence. The length of cDNA of rsbMx1, rsbMx2, rsbIL8, rbIL8 and rsbCOX2 genes are 2381bp, 2889bp, 865bp, 888bp and 2528bp respectively and that translates into 628, 637, 100, 99 and 608 amino acids respectively. The rsbMx1 and rsbMx2 genomic sequence were found to span 12 exons separated by 11 introns. The rsbIL8 and rbIL8 genomic sequence were found to span 4 exons separated by 3 introns. The rsbCOX-2 genomic sequence were found to span 10 exons separated by 9 introns. Also we studied if the experimental infection of red sea bream and rock bream with iridovirus could affect the expression of inflammatory cytokines, such as IL-8, COX-2 and IL-1β and an interferon-induced Mx genes. When injected with poly I:C, the Mx gene peak level of expressed immune genes in red sea bream and rock bream was day 1 post injection and the expression level of rsbMx1, rsbMx2 and rock bream Mx genes was similar about 22fold. While, when injected with iridovirus, the rsbMx1 and rsbMx2 genes reponse was peaking on day 3 and the expression level was about 88-fold and 208-fold respectively. However, rock bream Mx gene was peaking on day 7 and the level was about 33-fold compared to β-actin. But, inflammatory cytokines, such as IL-8, COX-2 and IL-1β genes expression was low compared with control. Cloned and analyzed various immune genes in rock bream and red sea bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention against viral infection.