红鳍东方鲀白介素15的原核表达及其活性测定  

Prokaryotic expression and biological activity evaluation of recombinan IL15 from tiger pufferfish Takifugu rubripes

作　　者：孙赛红 马普[1] 李慧[1] 孙鹤[1] 孔德荣[1] 李凤华 仇雪梅[1] 姜志强[1] 刘海映[3] 刘圣聪 王秀利[1] 

机构地区：[1]大连海洋大学水产与生命学院,辽宁大连116023 [2]大连三仪动物药品有限公司,辽宁大连116036 [3]大连海洋大学海洋科技与环境学院,辽宁大连116023 [4]大连天正实业有限公司,辽宁大连116011

出　　处：《大连海洋大学学报》2015年第6期598-603,共6页Journal of Dalian Ocean University

基　　金：国家海洋公益性行业科研专项(201405003-2);大连市科技计划项目(2014B11NC091)

摘　　要：为了开发红鳍东方鲀Takifugu rubripes免疫基因及其重组表达,生产重组免疫因子蛋白,通过提取红鳍东方鲀肾脏组织总RNA,利用RT-PCR技术扩增得到白介素15(interleukin15,IL15)基因序列,将IL15成熟肽序列与原核表达载体p ET-32a(+)连接,成功构建重组表达载体(重组子)p ET-32a(+)-IL15,并将其重组子转化至Escheria coli BL21(DE3)感受态细胞中获得重组表达IL15的基因工程菌,再经IPTG诱导,p ET-32a(+)-IL15在BL21中得到了融合表达。结果表明:通过对表达产物的纯化和Western Blotting检测,红鳍东方鲀IL15在大肠杆菌中的表达量较高,蛋白浓度为294.6μg/m L;用MTT法对该蛋白进行活性鉴定显示,加入稀释10倍和100倍的重组红鳍东方鲀IL15蛋白时,CTLL-2细胞的增殖率相对较高,分别为71%和73%。研究表明,重组红鳍东方鲀IL15蛋白具有体外促进CTLL-2细胞增值的能力。To exploit recomibinant protein of tiger pufferfish Takifugu rubripes by expression of immune gene and recominant immune factors,the tiger pufferfish interleukin-15（IL15） gene sequence was obtained from kidney tissue by RT-PCR,and the recombinant DNA p ET-32a（＋）-IL15 was constructed by link mature peptide gene sequences and prokaryotic expression vector p ET-32a（＋）.The genetic engineering bacteria of recombinant expression IL15 by transforming p ET-32a（＋）-IL15 into Escheria coli BL21（DE3）,and the fusion proteins were obtained under the induction of IPTG.The purification of the express products and Western Blotting test revealed that there was high-efficiency expression of recombinant DNA p ET-32a（＋）-IL15 in E.coli with the accurate target protein,and fusion protein concentration of 294.6 μg / m L.The evaluation of fusion protein activity by MTT method showed that the CTLL-2 cell proliferation rate was 71% and 73% at the diluted 10 times and 100 times fusion protein,respectively.The findings indicate that the recombinant IL15 fusion protein has the ability to promote the proliferation of CTLL-2 cells in vitro.

关 键 词：红鳍东方鲀 白介素-15 原核表达 活性鉴定 

分 类 号：S965.225[农业科学—水产养殖]