三角帆蚌基质金属蛋白酶基因的克

发布时间：2018-05-13 16:37

本文选题：三角帆蚌 + 基质金属蛋白酶　；参考：《南昌大学》2017年硕士论文

【摘要】：三角帆蚌(Hyriopsis cumingii)是我国重要的淡水育珠蚌之一,育珠插核过程中伤口容易受到病原体侵染,使育珠蚌的吐核率升高,甚至造成蚌的死亡。因此,开展对其分子免疫及伤口修复机制的研究具有重要的理论和实际意义。本文采用RACE PCR技术分别克隆出了三角帆蚌的基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶-19(MMP-19)的cDNA序列,利用实时荧光定量PCR方法检测2种基因在健康蚌血淋巴、肝胰脏、闭壳肌、外套膜和鳃5种组织中的表达情况;以及嗜水气单胞菌和肽聚糖(PGN)刺激后,2种基因在血液和肝胰脏中的表达变化;通过建立三角帆蚌创伤修复模型,利用实时荧光定量PCR方法检测2种基因在伤口修复期间不同时间段的表达情况;利用原核表达技术对2种基因进行了原核表达。MMP1基因的cDNA全长序列为1822 bp,其中5’UTR(非编码区)有31 bp;3’UTR有258 bp;开放阅读框(ORF)的碱基长度为1533 bp,共编码510个氨基酸。经Expasy在线网站的SignalP 4.0分析氨基酸发现该蛋白含有信号肽序列,该蛋白理论分子量为58.28 kDa,等电点为9.27。克隆得到的MMP19基因序列长度2130 bp,其中5’UTR 29 bp,3’UTR长度为532 bp,开放阅读框长度为1569 bp,共编码522氨基酸,经分析发现该蛋白含有信号肽序列。推导的蛋白分子量为59.44 kDa,等电点为6.83。MMP1,19基因的mRNA在健康蚌的血液、外套膜、肌肉、鳃和肝胰腺中均有表达。其中,MMP1和MMP19均在在血液中表达量最高。MMP1在嗜水气单胞菌刺激后,在24 h的血液和肝胰脏中均表达量极显著性上升;在PGN诱导后3 h的血液中极显著性上调,在肝胰脏中表达量无显著变化。MMP19经PGN和嗜水气单胞菌刺激后在肝脏中表达量变化不明显,在血液中经PGN诱导后MMP19表达量上调,在3 h表达量最高。经嗜水气单胞菌刺激后MMP19在血液中呈上调趋势,24 h表达量最高。MMP1基因在创伤后的第3 d表达量最高,MMP19基因在创伤后第1 d表达量最高,随着时间的增加,2种基因的表达量呈降低趋势,到达第15 d时表达量恢复到初始水平。将三角帆蚌MMP1,19基因的扩增产物和表达载体PET-28a利用BamHI、Hind III和EcoRI、HindIII双酶切之后,连接并成功构建了pET-28a-MMP1和pET-28a-MMP19原核表达重组质粒。将重组质粒转入大肠杆菌Rosetta-gami(DE3)中进行原核表达,经SDS-PAGE电泳检测,结果表明重组蛋白均以不溶的包涵体形式存在,纯化出的MMP19蛋白浓度为0.094 mg/m L。
[Abstract]:Hyriopsis cumingii (Hyriopsis cumingii) is one of the most important freshwater Hyriopsis cumingii in China. The wound of Hyriopsis cumingii is easy to be infected by pathogen during the process of bead implantation, which results in the increase of spitting rate of Hyriopsis cumingii and even the death of Hyriopsis cumingii. Therefore, it has important theoretical and practical significance to study its molecular immunity and wound repair mechanism. The cDNA sequences of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-19 (MMP-19) of mussel Hyriopsis cumingii were cloned by RACE PCR technique, and real-time fluorescence quantitative PCR was used to detect the two genes in the hemolymph, hepatopancreas and obturator muscle of healthy mussel. The expression changes of the two genes in blood and hepatopancreas after stimulation of Aeromonas hydrophila and peptidoglycan PGN. were established by establishing a wound repair model of Hyriopsis cumingii. The expression of two genes during wound repair was detected by real-time fluorescence quantitative PCR. The full-length cDNA sequence of prokaryotic expression. MMP1 gene of two genes was 1822 BP, of which 5 UTRs (non-coding region) had 31 BP / 3 UTR (258bp), and the length of open reading frame (ORF) was 1533 BP, encoding 510 amino acids. It was found that the protein contained signal peptide sequence by SignalP 4.0 analysis of Expasy online website. The theoretical molecular weight and isoelectric point of the protein were 58.28 kDa and 9.27 respectively. The length of the cloned MMP19 gene was 2130 BP, of which the length of the 5'UTR 29 bpt3 UTR was 532bpand the length of the open reading frame was 1569 BP, encoding 522 amino acids. It was found that the protein contained a signal peptide sequence. The deduced protein molecular weight was 59.44 kDa.The isoelectric point was 6.83.MMP1n19 gene mRNA was expressed in the blood, mantle, muscle, Gill and hepatopancreas of healthy mussel. After stimulation by Aeromonas hydrophila, the expression of MMP1 and MMP19 in the blood and hepatopancreas increased significantly at 24 h, and in the blood at 3 h after PGN induction, the expression of MMP1 and MMP1 increased significantly. There was no significant change in the expression of MMP19 in hepatopancreas. MMP19 was stimulated by PGN and Aeromonas hydrophila. The expression of MMP19 was not changed significantly in liver, but the expression of MMP19 was up-regulated in blood induced by PGN, and reached the highest level at 3 h. After stimulation by Aeromonas hydrophila, the expression of MMP19 in blood showed an up-regulation trend. The highest expression of MMP1 gene was observed on the 3rd day after trauma. The expression of MMP19 gene was the highest on the first day after trauma. With the increase of time, the expression of the two genes decreased, and the expression returned to the initial level at the 15th day. The amplification product of MMP1O19 gene and the expression vector PET-28a were digested by BamHIHind III and Ecor I HindIII, and the prokaryotic expression plasmids pET-28a-MMP1 and pET-28a-MMP19 were successfully constructed. The recombinant plasmid was transferred into E. coli Rosetta-gamide _ 3 for prokaryotic expression. The results of SDS-PAGE electrophoresis showed that the recombinant protein existed in the form of insoluble inclusion bodies, and the concentration of purified MMP19 protein was 0.094 mg/m / L.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S944.12

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