牦牛白细胞介素2(IL2)基因cDNA 的分子克隆和表达研究

摘要

Objective To study the immune genes and their immunological bioactivity of Yak. Method The experiment was conducted to clone of interleukin 2 gene (IL2) of yak. The cDNA of IL2 was firstly synthesized by RT-PCR from total RNA extracted from the Concanavalin A activated lymphocytes of yak blood and ligated into pMD-18 T vector and sequenced. The yak IL2 cDNA was then subcloned into pGEX 4T-1 and induced to express IL2 in prokaryotic cell by IPTG, which was analyzed by SDS-PAGE and the proliferation of lymphoblasts of yak. Result The sequencing analysis showed that a base mutation of C to T at position 442 of YIL2 coding region has produced a terminator (TAA), resulting in YIL2 protein to be shortened by 8 amino acids compared with other reported bovine IL2. Afterwards, the bioactivity of expression products of YIL2 cDNA was evaluated by detecting the proliferation of lymphoblasts of yak in vitro through MTT colorimetry, and the result indicated that this YIL2 protein could significantly promote the proliferation of Concanavalin A-stimulated lymphoblasts of yak. Conclusion The successful cloning and expression of yak IL2 gene is putatively useful for the development of novel immunopotentiator to raise the immunity of yak against various infectious pathogens and the immuno-protective efficacy of vaccinations.%目的克隆牦牛免疫基因并研究其免疫特性.方法以伴刀豆球蛋白A(Con A)激活的体外培养的牦牛血液淋巴细胞,提取激活淋巴细胞的总RNA, 用RT-PCR方法,从中克隆出白细胞介素2 cDNA,连接到T载体上测序,并亚克隆到pGEX 4T-1表达质粒, 在大肠杆菌进行了重组表达,纯化融合表达YIL2产物, MTT比色法测定其体外刺激牦牛血液淋巴母细胞增殖的免疫活性.结果 YIL2 cDNA序列分析显示:在其编码区442位点的一个碱基发生突变(由C突变为T),从而导致终止密码子(TAA)出现,使YIL2蛋白表达提前终止,与其它牛IL2蛋白相比少了8个氨基酸.MTT比色法测定结果表明重组牦牛IL2蛋白体外能显著促进牦牛淋巴母细胞的增殖.结论本实验成功克隆了牦牛IL2基因,其原核表达产物具有显著的免疫活性,这为研制新型免疫增强剂来提高牦牛对各种疾病的抵抗力,增强牦牛疫苗的免疫保护率奠定了基础.