Studies On Molecular Biology Of Innate Immune-related Factors Of The Crassostrea Ariakensis Gould Against RLO Infection

From late February2007, the oyster, Crassostrea ariakensis Gould, cultured in Guangxi province experienced mass mortalities over a large area and led to huge economic losses. A massive infection was found by an obligate intracellular prokaryote, a morphological specifically rickettsia-like organism (RLO). We investigated this RLO in the tissues of the oyster and carried out a series of researches about histopathology, ultrastructure, purification and chick embryo culture of this prokaryote.In order to get more information about the oyster immune defense mechanism against infection, a cDNA library of hemocyte from RLO challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in200unique sequences containing96known genes and104unknown genes. The96known genes were further categorized into11groups based on their biological process. Furthermore, eight genes including allograft inflammatory factor1(Ca-AIF1), high mobility group box protein (Ca-HMGB), three cyclophilin genes (Ca-CyPA, Ca-CyPB and Ca-PPIL3) and three complement-related fragments (CaC1q1,CaC1q2and CaC3) were selected. And sequence analyses and functional researches of them had been carried out as follows by immunology technology, molecular biotechnology, cell biology, bioinformatics methods and so on.1、Ca-AIF1encodes a protein with139amino acid sequences, containing a typical Ca2+binding EF-hand motif. Homology analysis revealed that the deduced amino acid sequences of Ca-AIFl share an identity from47%to73%with other AIF1s in various species. Through PET32a-AIF1constructed, we succeeded to express and purify Ca-AIF1in the E.coli BL21(DE3) cells and then the polyclonal antiserum against New Zealand white rabbits was prepared.The mRNA expression level of Ca-AIF1in various tissues was detected by real-time RT-PCR. Results showed that Ca-AIF1was ubiquitously expressed in all examined tissues and the expression levels in hemocyte, gills and gonads were higher than the others, suggesting that Ca-AIF1may involve in host immune response and germ cell proliferation and differentiation. Immunofluorescence with a laser confocal microscope was further performed to detect the cell localization of Ca-AIF1in gonads and the result showed that Ca-AIFl mainly localized in the epithelium cells.Real-time RT-PCR and Flow cytometer analyses were then taken to explore the function and mechanism of Ca-AIFl and its antiserum in the oyster immune defense response against infections. Results showed as follow (1) mRNA expression levels of Ca-AIF1were up-regulated after RLO/LPS stimulates, suggesting that Ca-AIF1may take part in the oyster immune response for anti-inflammation;(2) The mRNA expression levels of LITAF, Myd88and TGF β were significantly up-regulated using purified Ca-AIF1fusion protein to stimulate monolayer hemocytes, suggested that Ca-AIF1may act as a pro-inflammatory cytokine, and can induce the oyster immune response for anti-inflammation and possibly pass by Myd88-mediated signal pathway;(3) Administration of anti-CaAIF1serum significantly reduced the up-regulated rate of mRNA expression of LITAF which induced by RLO/LPS stimulation, indicating that anti-CaAIFl can significantly suppress RLO/LPS-induced inflammatory response;(4) Administration of anti-CaAIFl serum effectively reduced the rate of oyster hemocytes necrosis and apoptosis and increased cell viability, further indicating that anti-CaAIF1serum can effectively protect the oyster against Gram-negative bacterium and RLO infection2、Ca-HMGB encodes a protein with203amino acid sequences, containing two typical HMG-box domains and an acidic C-tails. Homology analysis revealed that the deduced amino acid sequences of Ca-HMGB share an identity from34%to55%with other HMGBs in various species. Through PET32a-HMGB constructed, we succeeded to express and purify Ca-HMGB in the E.coli Rosseta (DE3) cells, and then the polyclonal antiserum against New Zealand white rabbits was prepared.The mRNA expression levels of Ca-HMGB in various tissues were detected by real-time RT-PCR. Results showed that Ca-HMGB was ubiquitously expressed in all examined tissues and the expression level in hemocyte was significantly higher than the others, suggesting that Ca-HMGB may involve in host immune response. Immunofluorescence with a laser confocal microscope was further performed to detect the subcellular localization of Ca-HMGB in the hemocyte before and after RLO stimulation. Results showed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before stimulating, and12h after RLO stimulation, Ca-HMGB mostly localized in the cytoplasm of hemocytes.Real-time RT-PCR, western blot and Flow cytometer analyses were then taken to explore the function and mechanism of Ca-HMGB and its antiserum in the oyster immune defense response against infection. Results showed as follow (1) mRNA expression level of Ca-HMGB was up-regulated after RLO/LPS stimulation;(2) Ca-HMGB protein was released extracellular after RLO stimulation, taking part in the oyster immune response for anti-inflammation;(3) Except AIF1, the mRNA expression levels of cytokines LITAF, Myd88and TGF β were significantly up-regulated using purified Ca-HMGB fusion protein to stimulate monolayer hemocytes, suggesting that Ca-HMGB may act as a pro-inflammatory cytokine and can induce the oyster immune response for anti-inflammation and possibly pass by Myd88-mediated signal pathway;(4) Administration of anti-CaHMGB serum significantly reduced the up-regulated rate of mRNA expression of LITAF which induced by RLO/LPS stimulation, indicating that anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory response;(4) Administration of anti-CaHMGB serum effectively reduced the rate of oyster hemocytes necrosis and apoptosis and increased cell viability, further indicating that anti-CaHMGB serum can effectively protect the oyster against Gram-negative bacterium and RLO infection3、Ca-CyPA encodes a protein with165amino acid sequences. Ca-CyPB encodes a protein with217amino acid sequences and Ca-PPIL3encodes a protein with162amino acid sequences. All of the three cyclophilin genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Homology analysis revealed that the deduced amino acid sequences of three cyclophilin genes share identity with35%-48%. To other species, deduced amino acid sequence of Ca-CyPA share an identity from74-82%with other CyPA proteins, Ca-CyPB share an identity from57%～70%with other CyPB proteins and Ca-PPIL3share an identity from71%-79%with other PPIL3proteins.Through PET32a-CyPs constructed, we succeeded to express and purify Ca-CyPA, Ca-CyPB and Ca-PPIL3in the E.coli BL21(DE3) cells, and then the polyclonal antiserum against New Zealand white rabbits were prepared.Real-time RT-PCR was used to detect three cyclophilin genes tissue distribution and temporal expression profile in the hemocyte monolayers after RLO stimulation. Results showed that Ca-CyPA, Ca-CyPB and Ca-PPIL3were ubiquitously expressed in all examined tissues with the highest expression level in hemocyte. And mRNA expression levels of Ca-CyPA, Ca-CyPB and Ca-PPIL3were up-regulated after RLO stimulation, suggesting that Ca-CyPA, Ca-CyPB and Ca-PPIL3may take part in the oyster immune response for anti-inflammation.4、Among three complement-related fragments, deduced amino acid sequences of CaClq1and CaClq2contain a conversed Clq domain, belonging to the ClqDC family and CaC3contains conserved A2M_recep and C345C domain, belonging to C3-like proteins.Real-time RT-PCR was used to detect three complement-related fragments tissue distribution and temporal expression profile in the hemocyte monolayers after RLO stimulation. Results showed that CaClql, CaClq2and CaC3were ubiquitously expressed in all examined tissues with the highest expression level in hemocytes. And mRNA expression levels of them were up-regulated after RLO stimulation, suggesting that CaClq1, CaClq2and CaC3may take part in the oyster immune response.