三角帆蚌巨噬细胞移动抑制因子和Smad5基因克隆及表达

发布时间：2018-06-02 14:11

本文选题：三角帆蚌 + MIF　；参考：《南昌大学》2016年硕士论文

【摘要】：本文采用巢氏PCR方法和RACE PCR技术分别克隆出了三角帆蚌的巨噬细胞移动抑制因子(MIF)的cDNA序列和Smad5基因的cDNA序列,通过实时荧光定量PCR方法检测2个基因在蚌血液、肝胰脏、闭壳肌、外套膜和鳃等几种不同组织中的表达情况,通过肽聚糖、脂多糖、嗜水气单胞菌和葡聚糖对三角帆蚌的刺激,检测2个基因在血液和肝胰脏中的表达量的变化。克隆得到的MIF基因的cDNA全长序列为723 bp,其中5’UTR有69 bp;3’UTR有309 bp;开放阅读框的碱基长度为345 bp,342个碱基编码114个氨基酸。经Expasy在线网站的SignalP 4.1分析氨基酸,该蛋白理论分子量为13.83kD,等电点为5.91,不含信号肽。此氨基酸序列没有Trp残基,其结构平均疏水性为-0.165,一共有4个丝磷酸化位点,分别为Ser13,Ser59,Thr10,Thy37,没有硫化位点。三角帆蚌MIF蛋白的预测三级结构其均由6个α螺旋和12个β折叠片组成,MIF含有三个结构域,是一个三聚体蛋白质。利用实时定量PCR检测了MIF基因在三角帆蚌不同组织中的表达情况。结果表明MIF的mRNA在蚌的血液、外套膜、肌肉、鳃和肝胰腺各组织中均有表达。其中,肌肉组织的表达量最高,肝胰腺次之,外套膜和鳃的表达量相当,血液中最少。分别用嗜水气单胞菌和脂多糖刺激后,MIF的mRNA在血液和肝胰脏中均有一定量的表达,在肝胰脏中的变化最为明显,分别是空白组的2.1倍和1.19倍。结果表明三角帆蚌在受到病害后,MIF起着免疫防御的作用。将三角帆蚌MIF序列与表达载体PET-30a连接,成功构建了PET-30a-MIF原核表达质粒,利用大肠杆菌表达系统进行表达并纯化出蛋白。经分析确认重组蛋白20 KD左右,与预测的蛋白大小相符。Smad5基因序列长度1627 bp,其中5’UTR(非编码区)7 bp,开放阅读框(ORF)长度为1386 bp,3’UTR长度为234 bp,编码461个氨基酸,经分析发现该蛋白含有部分信号肽序列。推导的蛋白分子量为51 KD,等电点为7.27。推导的Smad5氨基酸序列中包含2个高度保守的Smad蛋白结构域MH1和MH2。
[Abstract]:The cDNA sequence of macrophage migration inhibitory factor (MIF) and the cDNA sequence of Smad5 gene were cloned by PCR and RACE PCR technique respectively. The two genes were detected in the blood and hepatopancreas by real-time fluorescence quantitative PCR method. The expression of two genes in blood and hepatopancreas was detected by stimulation of peptidoglycan, lipopolysaccharide, Aeromonas hydrophila and dextran. The full-length cDNA sequence of the cloned MIF gene was 723 BP, of which the 5'UTR was 69 BP / 3 UTR and the open reading frame was 345 BP / 342 nucleotide encoding 114 amino acids. The amino acid was analyzed by SignalP 4.1 of Expasy online website. The theoretical molecular weight of the protein was 13.83 KD, the isoelectric point was 5.91, and the protein contained no signal peptide. The amino acid sequence had no Trp residue, and its average hydrophobicity was -0.165. The amino acid sequence had four phosphorylation sites, which were Ser13N Ser59, Thr10, Thy37, and had no sulfidation sites. The predicted tertiary structure of MIF protein of Hyriopsis cumingii consists of 6 伪 helix and 12 尾 -fold sheets, which contains three domains and is a trimer protein. The expression of MIF gene in different tissues of Hyriopsis cumingii was detected by real time quantitative PCR. The results showed that MIF mRNA was expressed in blood, mantle, muscle, Gill and hepatopancreas of mussel. The highest expression was in muscle, followed by hepatopancreas, the same in mantle and gills, and the least in blood. After stimulation with Aeromonas hydrophila and lipopolysaccharide respectively, the expression of mRNA in blood and hepatopancreas was found in a certain amount, and the change was most obvious in hepatopancreas, 2.1 times and 1.19 times as much as that in the blank group, respectively. The results showed that MIF played an immune defense role in Hyriopsis cumingii. The MIF sequence of Hyriopsis cumingii was connected with the expression vector PET-30a, and the prokaryotic expression plasmid of PET-30a-MIF was constructed successfully. The expression system was used to express and purify the protein. It was confirmed by analysis that the recombinant protein was about 20KD, and the length of Smad5 gene was 1627 BP, in which the length of 5 UTRs was 1386 BP, and the length of ORFR was 1386 BP, encoding 461 amino acids, the length of the recombinant protein was about 20KD, and the length of Smad5 gene was 1627 BP, in which the length of 5 UTRs was 1386 BP, and the length of ORFR was 1386 BP. It was found that the protein contained partial signal peptide sequence. The deduced molecular weight of the protein is 51 KD and the isoelectric point is 7.27. The deduced Smad5 amino acid sequence contains two highly conserved Smad domains, MH1 and MH _ 2.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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