中国白兔白介素-15基因的克隆、序列分析及其在E.coli中的表达  

Cloning,sequence analysis of Chinese white rabbit IL-15 gene and its expression in E.coli

作　　者：孟庆玲[1] 才学鹏[1] 乔军[1] 骆学农[1] 景志忠[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省人兽共患病重点实验室

出　　处：《中国兽医学报》2008年第5期518-522,共5页Chinese Journal of Veterinary Science

基　　金：国家重大基础研究“973”资助项目(G1999011906)

摘　　要：以RT-PCR技术对用ConA刺激的中国白兔外周血淋巴细胞（PMBCr）进行扩增,将纯化后的PCR产物克隆入pMD18-T中进行核苷酸序列测定,并与不同物种的IL-15基因进行序列比较。结果IL-15基因全长489 bp,编码162个氨基酸,其中前29个氨基酸残基构成信号肽序列。与不同物种IL-15基因相比,核苷酸和推导的氨基酸序列有一定的差异。在推导的中国白兔IL-15氨基酸序列中,在108～110、119～121、127～129和143～146位存在4个潜在的N-联糖基化位点,同时存在6个Cys残基。将pTIL-15双酶切,回收目的基因片段克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-15,转化大肠杆菌BL21（DE3）,并用IPTG进行了诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为20 500的重组目的蛋白。经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的13.6%。Chinese white rabbit IL-15 gene was amplified by RT-PCR method from the PMBC stimulated by ConA. Then the positive PCR product was purified and ligatured with pMD18-T. The positive recombinant was used for sequencing. The complete length of white rabbit IL-15 gene was 489 bp, encoding 162 amino acids. Compared with the IL-15 genes of other species, there were some differences in the nucleotide sequence and deduced amino acid sequence. There were four N-glycosylation sites and 6 conservative Cys in the deduced amino acid sequence. Meanwhile,the IL-15 gene was subcloned into the prokaryotic expressing vector pET28a and transformed into host E. coli strain BL21 （DE3） for expression under the induction of IPTG. The expression of IL-15 protein was detected by SDS-PAGE. The results revealed that it had a molecular weight of 20 500,amounting to 13.6% in the total protein of the induced bacteria by the assaying of gel scanning.

关 键 词：兔白介素-15基因 克隆 序列分析 大肠杆菌表达 

分 类 号：Q78[生物学—分子生物学]