摘要
白细胞介素15(Interleukin-15,IL-15)是一个多功能细胞因子,在免疫系统和非免疫系统中都有重要作用,包括刺激T细胞增殖和激活自然杀伤细胞,可引起肌肉萎缩性刺激和肌肉收缩反应.但是,在山羊中该基因的研究尚未见报道.该研究采用RT-PCR方法克隆了山羊肌肉IL-15基因,使用荧光定量的方法构建该基因组织表达谱.序列分析表明山羊IL-15包含486bp全长编码序列,该序列编码162个氨基酸,可形成4-α-螺旋的空间结构,其核苷酸和氨基酸序列与绵羊(97.55%,94.48%)和牛(95.91%,92.02%)的同源性最高,与鸡的同源性最低(48.59%,27.89%).荧光定量检测表明该基因在肌肉和脾中相对表达量最高,在大脑中相对表达量最低.进一步通过PCR方法获得缺失长信号肽的成熟肽蛋白基因序列,重组到原核表达载体pET32a(+)中进行原核表达,获得29kDa的融合蛋白,并通过Western blot方法进行了鉴定.这些结果说明IL-15序列在哺乳动物间是保守的,在山羊中的功能与其他动物相似.成功构建的山羊IL-15表达系统为进一步研究山羊该基因的功能及应用奠定了基础.
Interleukin‐15 （IL‐15） is a pleiotropic cytokine involved in innate immunity as well as in muscle fiber protein synthesis .However ,there is no study of IL‐15 gene in goats .In this study ,IL‐15 gene was cloned from goat muscle tissue using the RT‐PCR method and the tissue expression pattern was evaluated using the fluorescence quantitative method .Sequences analysis showed that goat IL‐15 contained 486 bp ORF and encoded 162 amino acids forming a 4‐α‐helix .The nucleotide and amino acid sequence had high similarity with that of other animals ,especially with that of sheep （97.55% and 94.48% ） and cattle （95.91% and 92.02% ） .Quantification analysis showed that the gene had highest level of expression in muscle and spleen ,and lowest expression in brain .Furthermore ,a fusion protein of 29 kDa was expressed using the prokaryotic expression vector pET32a（＋ ） .The protein was further identified using the methods of western blot .In conclusion ,IL‐15 is conserved in mammals .Construction of IL‐15 expression system will lay a foundation for further study of its functions and application .
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第11期8-15,共8页
Journal of Southwest University(Natural Science Edition)
基金
重庆市自然科学基金计划重点项目(2009BA1052)
中央高校基本科研业务费专项(XDJK2011C022)
