【作者】 拜玲；

【导师】 潘和平；

【作者基本信息】 西北民族大学 ， 临床兽医， 2010， 硕士

【摘要】 论文以我国高原牦牛为研究对象,根据GenBank中收录的普通牛RPS5基因序列(登录号为XM-589792.3)设计一对特异性引物。通过RT-PCR技术从牦牛肝脏组织总RNA中扩增出RPS5基因的编码序列,将其连接至PMD18-T载体,转化JM109感受态细胞,获得阳性重组质粒;对其进行序列测定并利用分子生物学软件进行分析,同时构建RPS5蛋白物种进化树。将牦牛RPS5基因编码区连接至Pet28a表达载体,成功构建了原核表达载体Pet28a/ Rps5;通过IPTG诱导,摸索出了融合蛋白最佳表达条件。研究结果显示,经克隆获得牦牛RPS5基因1150 bp的cDNA序列,含615 bp的开放阅读框,编码204个氨基酸;对氨基酸序列进行信号肽分析。表明该蛋白质不存在信号肽,不属于分泌性蛋白质;与已知普通牛的RPS5基因编码区进行序列比对,发现存在1个碱基的变异,但未引起氨基酸的改变,不同物种RPS5蛋白有较高的保守性;RPS5蛋白物种进化树符合物种进化规律;SDS-PAGE电泳显示在27 kD处有特异性蛋白条带,表明牦牛RPS5基因成功实现了原核表达。更多还原

【Abstract】 This Gaoyuan Yak was takan as the candidates for the reasch. A pair of primers was designed and synthesized according to the bovine RPS5 gene sequence in Genbank (Accession Number: XM-589792.3). Coding sequence of RPS5 gene was amplified by RT-PCR technology from total RNA of yak’s liver, the product was cloned to PMD18-T vector and transfer into competent JM109-cells then got the positive recombinant; tested the sequence and analysed with molecular biology software. Phylogenetic tree of RPS5 protein was constructed. The coding region of Yak’s RPS5 gene was cloned to PET28A vector to construct an expression plasmid PET28A/ RPS5. After inducation with IPTG, it groped the best expression conditions of the fusion protein PET28A/ RPS5.The results showed that there was a 1150bp cDNA sequence of RPS5 gene cloned, including 615 bp open reading frame, encoding 204 amino acids. This protein is not secreted protein because we did not find singal peptide via the analysis of amino acid sequences Sequence alignment indicated that there were one mutations in the coding region between yak and bovine, but don’t cause amino acid mutation in RPS5 protein. RPS5 protein has high conservation in different species; phylogenetic tree conformed to the laws of species evolution. SDS-PAGE electrophoresis showed that the band with 27 kD of molecular weight was consistent as expected. The result indicated that the successful expression of RPS5 gene。更多还原