【作者】 邓传燕；

【导师】 吴灶和； 简纪常；

【作者基本信息】 广东海洋大学 ， 水产养殖， 2010， 硕士

【摘要】 本研究利用已构建的斜带石斑鱼(Epinephelus coioides)头肾Smart cDNA文库,首次克隆表达了斜带石斑鱼C-反应蛋白(EC-CRP)基因,并纯化获得了EC-CRP基因表达蛋白。EC-CRP的cDNA全长为946bp,其中5′非翻译区(5′-UTR)为148bp,3′非翻译区(3′-UTR)为123 bp,开放阅读框(ORF)为675bp,编码225aa。序列同源性分析发现,其氨基酸序列与大西洋鲑(Salmo salar)同源性最高,为42.58%。重组表达蛋白主要以包涵体形式存在,经亲和层析纯化获得带His标签的EC-CRP表达蛋白,SDS-PAGE电泳表明其分子量为25kDa。斜带石斑鱼CRP表达蛋白的获得为其疾病诊断和相关分子机理研究奠定了基础。Real-time PCR法研究溶藻弧菌免疫斜带石斑鱼后CRP基因在不同组织里的表达水平差异,结果发现,溶藻弧菌免疫斜带石斑鱼24h后CRP在斜带石斑鱼的头肾、脾脏、肝脏、肾脏、小肠、心脏、鳃、胸腺组织中均有表达,其中头肾中表达量最高,肝脏次之,然后依次是肾脏、脾脏、肠、心脏、和腮,胸腺中表达量最少。随着斜带石斑鱼感染时间的增长,CRP表达量在各组织达到峰值的时间不同:3h时鳃组织中表达量达峰值,为正常值的1.5倍;6h时胸腺组织中表达量达峰值,为正常值的2倍;12h时脾脏和肾脏组织中表达量达峰值,都为正常值的5倍;24h时头肾和肠组织中表达量达峰值,为正常表达量的50倍和1.5倍;48h时肝脏组织到达到峰值,为正常值的20倍。斜带石斑鱼在分别含10ppm苯酚、20ppm CdCl2和2ppm CuSO4的海水中暴露,以头肾组织为模式组织,发现随着时间的增加CRP表达量逐渐增加,分别在6h、6h、6h及12h处最高,分别为正常表达量的50倍、30倍和15倍。这些现象说明了鱼机体内对不同污染胁迫解毒紧迫性的必要程度可能有所差别。由于CRP的表达水平与暴露时间和污染物的性质有较好的关联性,而且随着暴露时间的延长与CRP浓度变化的趋势具有一致性,因此提示可以作为污染早期效应生物标志物。采用了溴化氰活化的Sepharose4B作为亲和吸附剂基质,选用phosphorylcholine chloride calcium作为配体,制得了纯化CRP的PC-Sepharose亲和层析柱,提取到了的CRP天然蛋白纯品。SDS-PAGE分析发现,该蛋白只含有一种亚基,大小约为26kDa,表明斜带石斑鱼和其他绝大部分鱼类一样,CRP中只含有一类亚基。本研究制备了兔抗CRP天然蛋白血清和兔抗CRP表达蛋白血清,发现前者能与斜带石斑鱼的血清发生抗原抗体反应,不能与CRP表达蛋白以及红笛鲷的血清发生抗原抗体反应;后者不能与CRP天然蛋白及斜带石斑鱼的血清发生抗原抗体反应,该抗体的制备为进一步研究C-反应蛋白的性质和功能奠定了基础。更多还原

【Abstract】 Based on the Smart cDNA libraries which constructed with head kidney of Epinephelus coioids, we cloned CRP gene. The cDNA length of CRP gene is 1001 bp,and includes a 148bp 5’-UTR, a 178bp 3’-UTR, a 675 bp ORF and encoding 225 amino acids. Sequence analysis reveals that EC-CRP gene shares a highest amino acids identity of 42.58% with Salmo salar. The CRP gene was cloned into the plasmid pET-21a(+) and expressed in Escherichia coli. The CRP gene was efficiently expressed in host bacterium E. coli BL21(DE3) following induction with IPTG. The expressed protease purified by Ni-NTA affinity chromatography. The SDS-PAGE analysis showed the recombinant EC-CRP was expressed as inclusion body,and the molecular weight was 25kDa. The results are important for the future study on molecular structure, function and diagnosis of disease-related genes of E. coioides.After injection of Vibrio alginolyticus, We found the concentration of CRP significant increasing in liver, head kidney, spleen, kidney, intestine, heart, gill and thymus of E. coioides by Real-time PCR. The highest concentration of CRP was in head kidney and liver second, following in kidney, spleen, intestines, heart and gills, and the lowest in thymus. As infection time increasing, the peak of expressed CRP was different in the organization at different times. The concentration of CRP was 1.5, 2, 5, 50, 1.5 and 20 times of the normal level in the gill at 3h, thymus at 6h, spleen at 12h, head kidney at 24h, intestinal at 24h and liver at 48h, respectively. The changes of expressed CRP were studied when E. coioids was exposed in the water containing CuSO4(2ppm), CdCl2(20ppm), phenol (10ppm), respectively. The maximum levels of CRP were15, 30 and 50 times higher than normal level at 6h. The results indicated that E. coioids was different in the detoxification extent under the different pollution stress. The amount of expressed CRP level associated with the exposure time of the natural pollutants. The results suggested that the natural pollution can be used as pollution biomarkers.The natural CRP had been purified by calcium dependent affinity chromatography with a phosphorylcholine (PC)–Sepharose column in the sera of E. coioides. SDS-PAGE analysis revealed that the protein contained only one kind of subunit and the molecular weight was 26kDa. In this study, rabbit antiserum against natural CRP and fusion CRP were prepared. Antigen-antibody reaction just occured between rabbit antiserum against natural CRP and the serum of E. coioides. rabbit antiserum against natural CRP couldn’t both react with the serum of Lutjanus sanguineus. rabbit The rabbit antiserum against fusion CRP had no reaction with the sera of E. coioides and nature CRP.更多还原