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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。
In order to develop the Takifugu rubripes immune gene and its recombinant expression, the recombinant immune protein was produced. The total RNA was extracted from the kidney tissues of the red tide of F. angustifolia by RT-PCR and the sequence of interleukin 15 (IL15) The recombinant expression vector p ET-32a (+) - IL15 was successfully constructed by ligating the IL15 mature peptide sequence with the prokaryotic expression vector p ET-32a (+) and the recombinant was transformed into Escherichia coli BL21 (DE3) After being induced by IPTG, pET-32a (+) - IL15 fusion protein was expressed in BL21. The results showed that the expression level of IL15 in Escherichia coli was 294.6μg / m L by purification of the expressed product and Western Blotting. The activity of IL15 was confirmed by MTT assay. CTLL-2 cells proliferated at a relatively high rate of 71% and 73%, respectively, when diluted 10-fold and 100-fold with recombinant IL-1. Studies have shown that the recombinant redfin Oriental flounder IL15 protein has the ability to promote the proliferation of CTLL-2 cells in vitro.