【作者】 张金洲；

【导师】 陈新华； 徐洵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 嗜水气单胞菌（Aeromonas hydrophila）是危害大黄鱼（Pseudosciana crocea）养殖的重要病原。本论文研究了大黄鱼内源性凋亡途径在细菌感染中的作用,为揭示内源性凋亡途径在抗细菌感染中的作用机制奠定了基础,也为进一步探索大黄鱼病害防治的新途径提供科学依据。通过简并引物、3’和5’RACE获得了大黄鱼内源性凋亡途径中的三个重要基因:cytochrome c（Lyccyt c）、caspase-9（Lyccasp9）和caspase-3（Lyccasp3）的全长cDNA。Lyccyt c编码104aa,其基因组含有2个外显子,1个内含子。Lyccasp9编码437 aa,包括三个结构域:Prodomain,90aa,包含CARD结构域;大亚基,133aa,包含组氨酸活性位点和半胱氨酸活性位点;小亚基,88aa。其基因组含有10个外显子,9个内含子。Lyccasp3编码285aa,包括两个结构域:大亚基,123aa,包含组氨酸活性位点和半胱氨酸活性位点;小亚基,共95aa。研究表明,重组Lyccasp9和Lyccasp3具有蛋白酶活性,证明它们具备caspase的基本性质。本实验中,我们首次在大黄鱼中对两个活性位点Lyccasp9的H²⁴⁹和C²⁹⁹及Lyccasp3的H¹³²和C¹⁷⁴进行了定点突变研究,其中对caspase组氨酸活性位点的定点突变研究在鱼类中未见报道。突变后的蛋白酶活性显著下降,证实了这两个残基的重要作用。RT-PCR分析显示,Lyccasp9和Lyccasp3基因在所检测的八个组织中为组成型表达。在Poly（I:C）或三联菌苗诱导下,Lyccasp9和Lyccasp3基因表达显著上调、进一步对Lyccasp9和Lyccasp3在组织中生物活性的测定,表明了内源性凋亡途径参与了大黄鱼的免疫过程。为了探明内源性凋亡途径在大黄鱼抗细菌感染中的作用,我们利用RNAi技术抑制了Lyccyt c和Lyccasp9的表达。通过Real-time PCR、Western blot、生物活性、TUNEL分析发现,Lyccyt c和Lyccasp9 dsRNA对大黄鱼的内源性凋亡途径有明显的抑制效果。在对Lyccyt c或Lyccasp9基因沉默后、感染嗜水气单胞菌条件下,这两个试验组的大黄鱼均在细菌感染后的4天内全部死亡,而注射GFP dsRNA和嗜水气单胞菌试验组及注射嗜水气单胞菌试验组均在细菌感染后的6天内全部死亡,注射PBS组死亡率只有20%左右。以上结果表明,在大黄鱼抗嗜水气单胞菌感染中、内源性凋亡途径引起的凋亡对大黄鱼有一定的保护作用。我们推测当嗜水气单胞菌侵入机体后、被细胞包裹,引起细胞凋亡,然后巨噬细胞进行吞噬,从而起到对机体的保护。同时还发现在内源性凋亡途径被抑制后,Lyccasp3基因的表达也受到了一定的抑制,进而抑制了细胞凋亡。另外还鉴定了大黄鱼两个新的CC型趋化因子,LycCC1和LycCC2。两者的序列一致性仅有17.7%,研究表明重组的LycCC1和LycCC2具有趋化活性。RT-PCR显示,LycCC1和LycCC2在所检测的9个组织中均为组成型表达。在Poly（I:C）或三联菌苗诱导下,LycCC1和LycCC2转录均明显上调、12h达到最高。我们发现Poly（I:C）对LycCC1的诱导比三联菌苗更为有效。研究表明,重组的LycCC1能诱导LMP10,MHC class Iαchain和β₂m的高表达。由此可见,LycCC1不仅参与了Poly（I:C）或三联菌苗诱导的免疫反应,还参与了大黄鱼MHC class I抗原呈递途径的调节。我们还发现三联菌苗对LycCC2的诱导比Poly（I:C）更为有效,这与LycCC1有明显不同,这预示LycCC2更侧重于细菌诱导的免疫反应。更多还原

【Abstract】 A. hydrophilais is one of the devastating pathogens in the farming of large yellow croaker. This study aimed at the mechanisms of intrinsic apoptotic pathway in large yellow croaker against the infection of A. hydrophilais. These works would be helpful for the enhancement of the ability against the diseases of large yellow croaker.In this investigation, we obtained three important genes, Cytochrome c（Lyccyt c）, caspase-9（Lyccasp9） and caspase-3（Lyccasp3） genes from large yellow croaker by degenerated primers, 3’ RACE and 5’ RACE.Lyccyt c gene encondes 104 aa. Genomic analysis reveals that it consists of ten exons and nine introns. Lyccasp9 gene encondes 437 aa, containing of three domains: Prodomain, 90 aa, which includes a CARD domain; P20, 133 aa, which includes His and Cys motif; P10, 88 aa. Lyccasp3 gene encondes 285 aa, consisting of two domains: P20,123 aa, which includes His and Cys motif; P10,95 aa.Experiment results indicated that rLyccasp9 and rLyccasp3 presented the enzyme activity of caspase. The recombinants Lyccasp9 with mutations of H²⁴⁹ to D or C²⁹⁹ to G, and Lyccasp3 with mutations of H¹³²to D or C¹⁷⁴ to G displayed decreased proteolytic activity, which confirmed their functional importance.It was showed by RT-PCR that Lyccasp9 and Lyccasp3 were constitutively expressed in all eight tissues examined. Time-course analysis using a real time PCR revealed that Lyccasp9 and Lyccasp3 transcripts in spleen and kidney were quickly increased in induction of Poly（I:C） or bacterial vaccine. This indicated that Lyccasp9 and Lyccasp3 participated in the immune of large yellow croaker. The bioactivity of Lyccasp9 and Lyccasp3 examined further confirmed the results.By analysis of Real-time PCR, Western blot, bioactivity and TUNEL, it was found that Lyccyt c dsRNA and Lyccasp9 dsRNA could markedly silence intrinsic apoptotic pathway. After inhibition of Lyccyt c and Lyccasp9 and injection of A. hydrophilais, the fish of the two group died in 4 d after infection, while the fish of the two group of injecting GFP and A. hydrophilais died in 6 d after infection, however the mortality of injecting PBS was about 20%. It was revealed that the apoptosis caused by intrinsic apoptotic pathway in large yellow croaker played positively role in the infection of A. hydrophilais. It was suggested that after infection upon large yellow croaker, A. hydrophilais is internalized by cells, then results in their apoptosis, which forms apoptotic body and induces marophages to lick up apoptotic body. Thus it contributes to the host defense against A. hydrophilais. It was also found that Lyccasp3 expression was inhibited to a certain extent after silencing of intrinsic apoptotic pathway in large yellow croaker, and apoptosis was also inhibited.Two novel CC chemokine genes were isolated from large yellow croaker, LycCC1 and LycCC2. LycCC1 and LycCC2 only have 17.7% aa sequence identities. Recombinant LycCC1 and LycCC2 protein produced in Pichia pastoris exhibited marked chemotaxis to the peripheral blood leucocytes （PBLs） from large yellow croaker. Tissue expression profile analysis showed that the two genes were constitutively expressed in all nine tissues examined. Upon stimulation with poly（I:C） or bacterial vaccine, LycCC1 gene expression was obviously up-regulated and reached their peak levels at 12 h post-induction. LycCC1 gene expression was more strongly up-regulated by poly（I:C） than by bacterial vaccine. An in vivo administration of rLycCC1 could significantly up-regulate the expression levels of LMP10, MHC class Iαchain andβ₂m. These results suggest that LycCC1 may not only have a pro-inflammatory function in immune response, but also be involved in adaptive immune response by modulating MHC class I antigen processing. However, LycCC2 expression was more strongly up-regulated by bacterial vaccine than by poly（I:C）. These results suggested that LycCC2 represent a novel member of fish CC chemokine family, involving in a pro-inflammatory function in immune response triggered by more bacterial vaccine than poly（I:C） in large yellow croaker.更多还原