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1、 褶纹冠蚌论文：褶纹冠蚌过氧化氢酶基因克隆、原核表达褶纹冠蚌论文：褶纹冠蚌过氧化氢酶基因克隆、原核表达及酶活性分析及酶活性分析【中文摘要】褶纹冠蚌(Crist aria plicata)是我国重要“淡水育珠蚌”之一,近年来褶纹冠蚌频繁受到病害的影响,对其育珠能力产生了极大的影响。本文对褶纹冠蚌过氧化氢酶(cpCAT)的 cDNA进行了克隆与原核表达。本文利用 RACE-PCR 及同源克隆方法,克隆出 cpCAT cDNA 全长。该基因全长为 4863 bp,且有 3 个外显子,2 个内含子。cpCAT cDNA 全长为 2618 bp,其中编码区 1503 bp,5端不翻译区 136 bp,3
2、端不翻译区 979 bp,具有典型的腺苷酸信号序列AATAAA 和 PolyA 尾。该基因序列开放阅读框编码为 501 个氨基酸,预测蛋白质分子量为 56.86kDa,等电点为 6.77。BLAST 分析显示cpCAT 氨基酸序列和动物,植物,细菌的 CAT 基因有很高的同源性。cpCAT 无信号肽,存在过氧化氢酶近端活性位点(61FNRERIPERVVHAKGAG77)和过氧化氢酶近端血红素配体签名序列(351RLYSYSDTH359),两个糖基化位点(145NNTP148)与(436NFSQ439)。系统发育树结果表明褶纹冠蚌与栉孔扇贝(Chlamys farreri)过氧化氢酶基因的亲缘
3、关系最近。利用 RT-PCR 分析检测 cpCAT 基因经注射嗜水气单胞菌后的表达情况,结果表明在注射嗜水气单胞菌后,在血液、鳃、外套膜、闭壳肌和肝脏中都有表达,且在肝脏中的表达量最高,且各个组织中 cpCAT 基因的表达明显增加,12h 后达到最大值,随后表达有所下降,到 48h 后恢复至原有水平。结果表明褶纹冠蚌在受到病害后,其体内的过氧化氢酶具有防御作用。根据开放阅读框设计带酶切位点(KpnI 和 EcoRI)的引物,构建重组表达质粒,经 IPTG 诱导表达,利用 SDS-PAGE 分析表达产物。结果表明重组 cpCAT 在大肠杆菌(Escherichia coli) Rosetta-g
4、ami (DE3)中获得了表达,产物为56.86 KDa。纯化的 cpCAT 的酶活力可达 11194.4+40.4U/mg,该酶最适温度为 25,有较高的热稳定性(60以下),cpCAT 的 pH 值的最适范围在 pH5.0-10.0,最适 pH 值为 7.0。用变性剂 Urea 和 SDS 处理时酶活性有所改变,用 1%3%SDS 和 24mol/L Urea 处理时,酶活性保持 80%以上。随着变性剂浓度的增加,cpCAT 酶活力逐渐降低。【英文摘要】Cristaria plicala is one of the most important “Freshwater pearl muss
5、el” in our country. But recently Cristaria plicata is vulnerable to disease frequently, which makes a great influence to the ability of the pearl. The catalase cDNA of C. plicata (cpCAT) was cloned and the full length was expressed with Prokaryotic expression in the paper.The cpCAT cDNA full sequenc
6、e has been cloned using homologous cloning and RACE-PCR. The gene is 4863 bp long and has a total of two introns and three exons. In addition the full-length cDNA of cpCAT contained 2618 bp, The cDNA contained a 5untranslated region (UTR) of 136 nucleotides, the 3UTR of 979 bp with a canonical polya
7、denylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1503 bp, encoding 501 amino acid residues with 56.86 kDa predicted molecular weight. The theoretical isoelectric point was 6.77. BLAST analysis showed that the deduced amino acid sequence of cpCAT had significant
8、homology to catalases from animals, plants and bacteria. The deduced amino acid sequence of cpCAT had characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77).heme-ligand signature motif (351RLYSYSDTH359), two glycosylation sites (N145, N436), NADPH binding sit
9、e and the three catalytic amino acid residues (His72, Asnl45 and Tyr355). It had no signal peptide. The phylogenetic tree indicated that cpCAT gene was very close to the gene of scallops, Chlamysfarreri.Expression of cpCAT after injected by Aeromonas hydrophila was determined by RT-PCR. The results
10、showed that after injected by A. hydrophila. The expression of cpCAT in different tissues of Cristaria plicata were determined by RT-PCR and the results showed the expression of cpCAT were detected in mantel, conductor muscle, hepatopancreas, sexual gland and hemocyte.and the best expression in hepa
11、topancreas. The results showed that after injected by A. hydrophila, the expression of cpCAT were increased in all the tissues, the expression of cpCAT were increased at first 12h, and decreased thereafter, however recovery the original level at 48h, which proved the cpCAT was important immune syste
12、m in C. plicata. According to the ORF, designed site (KpnI and EcoRI) primers, the constructed recombinant expression plasmids were induced by the chemical inducer IPTG. The expression products were analyzed by SDS-PAGE. The results indicated that recombination cpCAT was successfully expressed in Es
13、cherichia Coli. The expression of protein was 56.86 KDa. The enzymatic activity of purified recombinant cpCAT was 11194.440.4 U/mg, it might resist against H2O2.The recombinant enzyme holded higher thermal stability, the optimum temperature was 25, The stability of the recombinant enzyme were higher
14、 between pH 5 and 10, and the optimal pH value was 7.0. when cpCAT was treated with 2-4moL/L urea and 1%3% SDS, the activity was also stable, it kept more than 80% activity. With the increasing of the concentration of denaturation agent, the activity of cpCAT reduced gradually.【关键词】褶纹冠蚌 过氧化氢酶 基因克隆 原
15、核表达 酶活性【英文关键词】Cristaria plicata Catalase Gene cloning Prokaryotic expression Enzymatic activity【目录】褶纹冠蚌过氧化氢酶基因克隆、原核表达及酶活性分析 中文摘要 3-4 ABSTRACT 4-5 缩略语表 6-7 目录 7-9 第一章 文献综述 9-16 1 贝类的免疫学研究 9-13 1.1 贝类的细胞免疫 9-10 1.2 贝类的体液免疫 10-13 1.2.1 氧化酶类和抗氧化酶类 10-11 1.2.2 水解酶类 11 1.2.3 抗菌肽 11 1.2.4 凝集素 11-12 1.2.5 硝酸盐超氧化物阴离子 12 1.2.6 溶血素 12-13 2 过氧化氢酶基因 13-14 2.1 CAT 基因的种类和分布 13 2.2 CAT 基因结构特点和功能 13-14 2.3 CAT 基因的应用 14 2.3.1 CAT 基因在纺织方面的应用 14 2.3.2 CAT 基因在食品方面的应用 14 3 本研究的意义 14-16 第二章 褶纹冠蚌过氧化氢酶基因的克隆、原核表达及酶活性分析 16-47 1 前言 16 2 材料与方法 16-25 2.1 主要仪器设备 16-17 2.2 主要试剂 17 2.3 试验材料 17
