本文選題：巴什拜羊 + 湖羊　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：綿羊支原體肺炎是一種能引發(fā)綿羊以咳嗽、喘氣、漸進(jìn)性消瘦及肺間質(zhì)的增生性炎癥為特征的慢性呼吸道疾病,對(duì)養(yǎng)羊業(yè)危害嚴(yán)重。短的上腭、肺及鼻咽上皮克隆基因1(short palate,lung and nasal epithelium clone 1,SPLUNC1)是一種在呼吸道上皮高表達(dá)的蛋白質(zhì)分子,參與了宿主防御活動(dòng),具有抗菌和抗炎的功能,同時(shí)還能抑制肺炎支原體生長(zhǎng)。目的:本研究通過(guò)克隆巴什拜羊與湖羊的SPLUNC1基因c DNA全長(zhǎng)序列,對(duì)其進(jìn)行生物信息學(xué)分析,通過(guò)真核表達(dá)巴什拜羊與湖羊的SPLUNC1基因,研究重組SPLUNC1蛋白對(duì)綿羊肺炎支原體的作用,驗(yàn)證其生物活性,為下一步研究綿羊SPLUNC1蛋白的生物學(xué)功能奠定基礎(chǔ)。方法:(1)巴什拜羊與湖羊的SPLUNC1基因全長(zhǎng)c DNA的擴(kuò)增:根據(jù)Gen Bank上已報(bào)道的牛的SPLUNC1基因序列,設(shè)計(jì)引物,使用RACE技術(shù)分別克隆巴什拜羊與湖羊的SPLUNC1基因的3’端序列和5’端序列,并測(cè)序、拼接獲得巴什拜羊與湖羊的SPLUNC1基因的全長(zhǎng)c DNA序列。(2)巴什拜羊與湖羊的SPLUNC1基因全長(zhǎng)c DNA的生物信息學(xué)分析:利用生物信息學(xué)軟件及在線分析工具對(duì)獲得的巴什拜羊與湖羊SPLUNC1基因全長(zhǎng)c DNA序列進(jìn)行核酸及其編碼蛋白信號(hào)肽、亞細(xì)胞定位、二級(jí)結(jié)構(gòu)、三級(jí)結(jié)構(gòu)、進(jìn)化樹(shù)等生物信息學(xué)分析。(3)巴什拜羊與湖羊的SPLUNC1基因的克隆與表達(dá):使用PCR方法擴(kuò)增出巴什拜羊與湖羊的SPLUNC1基因的開(kāi)放閱讀框序列,并將該序列連接至p PIC9K真核表達(dá)載體,構(gòu)建p PIC9K-SPLUNC1表達(dá)質(zhì)粒,轉(zhuǎn)化至巴斯德畢赤酵母GS115感受態(tài)細(xì)胞中進(jìn)行誘導(dǎo)表達(dá),優(yōu)化誘導(dǎo)表達(dá)條件并對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE和Western Blotting分析鑒定。(4)重組巴什拜羊與湖羊的SPLUNC1蛋白的純化與生物活性研究:利用Ni柱純化方法分別對(duì)重組巴什拜羊與湖羊SPLUNC1蛋白進(jìn)行純化,將純化的蛋白作用于綿羊肺炎支原體,使用熒光實(shí)時(shí)定量法檢測(cè)純化蛋白對(duì)綿羊肺炎支原體的作用。結(jié)果與結(jié)論:(1)巴什拜羊、湖羊SPLUNC1基因全長(zhǎng)分別為1095bp和1091bp,核苷酸相似性為99%。(2)巴什拜羊、湖羊SPLUNC1的核苷酸序列有五處核苷酸堿基差異,但是二者的開(kāi)放閱讀框均為768bp,編碼氨基酸相同,均為255個(gè)氨基酸。SPLUNC1蛋白質(zhì)的分子量為26.53 KD,理論等電點(diǎn)為5.07。SPLUNC1蛋白N端存在信號(hào)肽,亞細(xì)胞定位在細(xì)胞外。SPLUNC1蛋白質(zhì)的二級(jí)結(jié)構(gòu)主要為α螺旋和無(wú)規(guī)則卷曲。該蛋白由4股反平行的肽段形成的β折疊片和2個(gè)α螺旋組成的桶型結(jié)構(gòu)域組成。系統(tǒng)發(fā)育樹(shù)分析結(jié)果說(shuō)明,SPLUNC1蛋白與山羊首先聚為一類(lèi),后與牛聚為一類(lèi),這與動(dòng)物學(xué)分類(lèi)結(jié)果一致。(3)在巴斯德畢赤酵母GS115中成功表達(dá)巴什拜羊與湖羊重組SPLUNC1蛋白,經(jīng)SDS-PAGE檢測(cè)發(fā)現(xiàn),蛋白的分子量均為25.53k D,且在甲醇誘導(dǎo)72h表達(dá)的蛋白含量較高,經(jīng)Western Blotting檢測(cè)證實(shí)表達(dá)蛋白為目的蛋白。(4)SDS-PAGE檢測(cè)純化的重組蛋白,結(jié)果顯示為單一的條帶,且分子量為25.53k D,說(shuō)明成功純化了目的蛋白。實(shí)時(shí)熒光定量PCR結(jié)果表明,巴什拜羊和湖羊的重組SPLUNC1蛋白對(duì)支原體生長(zhǎng)具有明顯的抑制作用,說(shuō)明表達(dá)的巴什拜羊和湖羊的SPLUNC1蛋白具有良好的生物學(xué)活性。
[Abstract]:Mycoplasma pneumoniae (MP) is a chronic respiratory disease characterized by cough, gasping, progressive emaciation and proliferative inflammation in the interstitial lung of the sheep. It is very harmful to the sheep industry. The short palate, lung and nasopharyngeal epithelial clone 1 (short palate, lung and nasal epithelium clone 1, SPLUNC1) is a high surface of the epithelium in the respiratory tract The protein molecule, which is involved in the host defense, has the function of antiseptic and anti-inflammatory, and also inhibits the growth of Mycoplasma pneumoniae. Objective: To study the bioinformatics analysis of the SPLUNC1 gene C DNA of bashbai sheep and Hu sheep, and to express the SPLUNC1 gene of bahbai sheep and Hu sheep through the eukaryotic expression. To investigate the effect of recombinant SPLUNC1 protein on Mycoplasma sheeppneumoniae and verify its biological activity, it lays the foundation for the next step of studying the biological function of sheep SPLUNC1 protein. Method: (1) amplification of the full length C DNA of the SPLUNC1 gene of basbai sheep and Hu sheep: design primers according to the SPLUNC1 gene sequence of cattle that have been reported on Gen Bank and use RACE Technology The 3 'end sequence and the 5' end sequence of the SPLUNC1 gene of bashbai sheep and Hu sheep were cloned and sequenced to obtain the full length C DNA sequence of the SPLUNC1 gene of bashbai sheep and Hu sheep. (2) the bioinformatics analysis of the full length C DNA of the SPLUNC1 gene of basbai sheep and Hu sheep: obtained by using bioinformatics software and online analysis tools. The full length C DNA sequence of basbai sheep and Hu sheep SPLUNC1 gene sequences nucleic acid and its encoded protein signal peptide, subcellular location, two stage structure, three grade structure, evolutionary tree and other bioinformatics analysis. (3) the cloning and expression of SPLUNC1 gene of basbai sheep and Hu sheep: the open reading of the SPLUNC1 gene of bahbai sheep and Hu sheep using PCR method Read frame sequence and connect the sequence to P PIC9K eukaryotic expression vector, construct P PIC9K-SPLUNC1 expression plasmid, transform it into GS115 receptive cells of Pichia pastoris to induce expression, optimize the induced expression conditions and identify the expression products by SDS-PAGE and Western Blotting analysis. (4) recombination of bashbai sheep and lake sheep SPLUNC1 Purification and biological activity of protein purification: the recombinant bahbai sheep and SPLUNC1 protein of Hu sheep were purified by Ni column purification method, the purified protein was acted on Mycoplasma pneumoniae, and the effect of the purified protein on Mycoplasma pneumoniae was detected by real time fluorescence quantitative method. The results and conclusions were as follows: (1) basbai sheep and Hu sheep SPLUNC1 base The nucleotide similarity is 99%. (2) basbai sheep and the nucleotide sequence of SPLUNC1 in Hu sheep has five nucleoside base differences, but the two open reading frame is 768bp, the encoding amino acid is the same, the molecular weight of the 255 amino acid.SPLUNC1 protein is 26.53 KD, and the theoretical isoelectric point is 5.07.SPLUNC1 protein. There is a signal peptide in the N terminal. The two level of subcellular localization of.SPLUNC1 protein is mainly alpha helix and irregular curl. This protein consists of 4 anti parallel peptide segments and 2 alpha helix structure domains. Phylogenetic tree analysis shows that the SPLUNC1 protein is first clustered with the goat and then with the cattle. The results were in accordance with the classification results of zoology. (3) the recombinant SPLUNC1 protein of bahiba sheep and lake sheep was successfully expressed in Pichia pastoris GS115 GS115. The molecular weight of the protein was found to be 25.53k D by SDS-PAGE detection, and the protein content expressed in the methanol induced 72h was higher, and the expression protein was confirmed by Western Blotting as the target protein. (4) the recombinant protein of the purified protein was detected by SDS-PAGE. The results showed a single band and the molecular weight of 25.53k D, indicating that the target protein was successfully purified. Real time fluorescence quantitative PCR results showed that the recombinant SPLUNC1 protein of basbai sheep and Hu sheep has an obvious inhibition effect on the growth of mycoplasma, indicating the SPLUNC1 of the expressed bahbai sheep and the lake sheep. Protein has good biological activity.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S858.26;Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 周后德;武明花;石磊;周鳴;楊一新;趙瑾;鄧坦;李小玲;沈守榮;李桂源;;分泌性蛋白SPLUNC1對(duì)上呼吸道綠膿桿菌的抑制作用[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2006年04期

2 陳啟龍;;RACE技術(shù)的研究進(jìn)展及其應(yīng)用[J];黃山學(xué)院學(xué)報(bào);2006年03期

3 徐燁;劉雅婷;代文瓊;朱曉媛;陳雪嬌;仵發(fā)靜;智龍;王曼君;;幾種主要的RACE技術(shù)及應(yīng)用[J];中國(guó)農(nóng)業(yè)科技導(dǎo)報(bào);2012年02期

相關(guān)博士學(xué)位論文 前1條

1 寧方坤;人源天然免疫相關(guān)蛋白SPLUNC1的結(jié)構(gòu)生物學(xué)研究[D];中國(guó)科學(xué)技術(shù)大學(xué);2012年

，

本文編號(hào)：1782717