绵羊SPLUNC1基因的克

发布时间：2018-04-21 13:53

本文选题：巴什拜羊 + 湖羊　；参考：《石河子大学》2015年硕士论文

【摘要】：绵羊支原体肺炎是一种能引发绵羊以咳嗽、喘气、渐进性消瘦及肺间质的增生性炎症为特征的慢性呼吸道疾病,对养羊业危害严重。短的上腭、肺及鼻咽上皮克隆基因1(short palate,lung and nasal epithelium clone 1,SPLUNC1)是一种在呼吸道上皮高表达的蛋白质分子,参与了宿主防御活动,具有抗菌和抗炎的功能,同时还能抑制肺炎支原体生长。目的:本研究通过克隆巴什拜羊与湖羊的SPLUNC1基因c DNA全长序列,对其进行生物信息学分析,通过真核表达巴什拜羊与湖羊的SPLUNC1基因,研究重组SPLUNC1蛋白对绵羊肺炎支原体的作用,验证其生物活性,为下一步研究绵羊SPLUNC1蛋白的生物学功能奠定基础。方法:(1)巴什拜羊与湖羊的SPLUNC1基因全长c DNA的扩增:根据Gen Bank上已报道的牛的SPLUNC1基因序列,设计引物,使用RACE技术分别克隆巴什拜羊与湖羊的SPLUNC1基因的3’端序列和5’端序列,并测序、拼接获得巴什拜羊与湖羊的SPLUNC1基因的全长c DNA序列。(2)巴什拜羊与湖羊的SPLUNC1基因全长c DNA的生物信息学分析:利用生物信息学软件及在线分析工具对获得的巴什拜羊与湖羊SPLUNC1基因全长c DNA序列进行核酸及其编码蛋白信号肽、亚细胞定位、二级结构、三级结构、进化树等生物信息学分析。(3)巴什拜羊与湖羊的SPLUNC1基因的克隆与表达:使用PCR方法扩增出巴什拜羊与湖羊的SPLUNC1基因的开放阅读框序列,并将该序列连接至p PIC9K真核表达载体,构建p PIC9K-SPLUNC1表达质粒,转化至巴斯德毕赤酵母GS115感受态细胞中进行诱导表达,优化诱导表达条件并对表达产物进行SDS-PAGE和Western Blotting分析鉴定。(4)重组巴什拜羊与湖羊的SPLUNC1蛋白的纯化与生物活性研究:利用Ni柱纯化方法分别对重组巴什拜羊与湖羊SPLUNC1蛋白进行纯化,将纯化的蛋白作用于绵羊肺炎支原体,使用荧光实时定量法检测纯化蛋白对绵羊肺炎支原体的作用。结果与结论:(1)巴什拜羊、湖羊SPLUNC1基因全长分别为1095bp和1091bp,核苷酸相似性为99%。(2)巴什拜羊、湖羊SPLUNC1的核苷酸序列有五处核苷酸碱基差异,但是二者的开放阅读框均为768bp,编码氨基酸相同,均为255个氨基酸。SPLUNC1蛋白质的分子量为26.53 KD,理论等电点为5.07。SPLUNC1蛋白N端存在信号肽,亚细胞定位在细胞外。SPLUNC1蛋白质的二级结构主要为α螺旋和无规则卷曲。该蛋白由4股反平行的肽段形成的β折叠片和2个α螺旋组成的桶型结构域组成。系统发育树分析结果说明,SPLUNC1蛋白与山羊首先聚为一类,后与牛聚为一类,这与动物学分类结果一致。(3)在巴斯德毕赤酵母GS115中成功表达巴什拜羊与湖羊重组SPLUNC1蛋白,经SDS-PAGE检测发现,蛋白的分子量均为25.53k D,且在甲醇诱导72h表达的蛋白含量较高,经Western Blotting检测证实表达蛋白为目的蛋白。(4)SDS-PAGE检测纯化的重组蛋白,结果显示为单一的条带,且分子量为25.53k D,说明成功纯化了目的蛋白。实时荧光定量PCR结果表明,巴什拜羊和湖羊的重组SPLUNC1蛋白对支原体生长具有明显的抑制作用,说明表达的巴什拜羊和湖羊的SPLUNC1蛋白具有良好的生物学活性。
[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.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.26;Q78

【参考文献】