本文選題：三角帆蚌 + MIF��； 參考：《南昌大學(xué)》2016年碩士論文

【摘要】：本文采用巢氏PCR方法和RACE PCR技術(shù)分別克隆出了三角帆蚌的巨噬細(xì)胞移動(dòng)抑制因子(MIF)的cDNA序列和Smad5基因的cDNA序列,通過(guò)實(shí)時(shí)熒光定量PCR方法檢測(cè)2個(gè)基因在蚌血液、肝胰臟、閉殼肌、外套膜和鰓等幾種不同組織中的表達(dá)情況,通過(guò)肽聚糖、脂多糖、嗜水氣單胞菌和葡聚糖對(duì)三角帆蚌的刺激,檢測(cè)2個(gè)基因在血液和肝胰臟中的表達(dá)量的變化。克隆得到的MIF基因的cDNA全長(zhǎng)序列為723 bp,其中5’UTR有69 bp;3’UTR有309 bp;開(kāi)放閱讀框的堿基長(zhǎng)度為345 bp,342個(gè)堿基編碼114個(gè)氨基酸。經(jīng)Expasy在線網(wǎng)站的SignalP 4.1分析氨基酸,該蛋白理論分子量為13.83kD,等電點(diǎn)為5.91,不含信號(hào)肽。此氨基酸序列沒(méi)有Trp殘基,其結(jié)構(gòu)平均疏水性為-0.165,一共有4個(gè)絲磷酸化位點(diǎn),分別為Ser13,Ser59,Thr10,Thy37,沒(méi)有硫化位點(diǎn)。三角帆蚌MIF蛋白的預(yù)測(cè)三級(jí)結(jié)構(gòu)其均由6個(gè)α螺旋和12個(gè)β折疊片組成,MIF含有三個(gè)結(jié)構(gòu)域,是一個(gè)三聚體蛋白質(zhì)。利用實(shí)時(shí)定量PCR檢測(cè)了MIF基因在三角帆蚌不同組織中的表達(dá)情況。結(jié)果表明MIF的mRNA在蚌的血液、外套膜、肌肉、鰓和肝胰腺各組織中均有表達(dá)。其中,肌肉組織的表達(dá)量最高,肝胰腺次之,外套膜和鰓的表達(dá)量相當(dāng),血液中最少。分別用嗜水氣單胞菌和脂多糖刺激后,MIF的mRNA在血液和肝胰臟中均有一定量的表達(dá),在肝胰臟中的變化最為明顯,分別是空白組的2.1倍和1.19倍。結(jié)果表明三角帆蚌在受到病害后,MIF起著免疫防御的作用。將三角帆蚌MIF序列與表達(dá)載體PET-30a連接,成功構(gòu)建了PET-30a-MIF原核表達(dá)質(zhì)粒,利用大腸桿菌表達(dá)系統(tǒng)進(jìn)行表達(dá)并純化出蛋白。經(jīng)分析確認(rèn)重組蛋白20 KD左右,與預(yù)測(cè)的蛋白大小相符。Smad5基因序列長(zhǎng)度1627 bp,其中5’UTR(非編碼區(qū))7 bp,開(kāi)放閱讀框(ORF)長(zhǎng)度為1386 bp,3’UTR長(zhǎng)度為234 bp,編碼461個(gè)氨基酸,經(jīng)分析發(fā)現(xiàn)該蛋白含有部分信號(hào)肽序列。推導(dǎo)的蛋白分子量為51 KD,等電點(diǎn)為7.27。推導(dǎo)的Smad5氨基酸序列中包含2個(gè)高度保守的Smad蛋白結(jié)構(gòu)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 偽 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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S917.4

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