水貂白细胞介素-4基因的克隆、序列分析与原核表达

为了获得高纯度的白细胞介素-4（IL-4）蛋白，对分离得到的水貂外周血淋巴细胞经植物血凝素（PHA）诱导后，提取淋巴细胞总RNA，根据GenBank中登录的雪貂IL-4基因序列，设计并合成特异性引物，通过RT-PCR扩增获得了水貂IL-4基因序列全长399 bp，编码132个氨基酸，与雪貂氨基酸序列同源性高达99.2%，与熊猫、犬同源性均为90.0%。将编码成熟蛋白基因构建到原核表达载体pProEX-HTb，IPTG诱导后，SDS-PAGE及Western blotting结果显示，IL-4表达产物为15 ku的包涵体蛋白。通过His-Trap HP亲和层析预装柱变性、复性洗脱可获得高纯度的重组IL-4蛋白。本试验为水貂IL-4基因的进一步研究奠定了基础。

Cloning,Sequence Analysis and Prokaryotic Expression of Mink Interleukin-4 Gene

In order to obtain high purity of interleukin 4 (IL-4) protein,the specific primers were designed and synthesized according to the mink IL-4 gene sequence in GenBank. The IL-4 gene was amplified by RT-PCR using total RNA of the lymphocyte isolated from the peripheral blood that induced by phytohemagglutinin (PHA). The length of IL-4 gene sequence was 399 bp which encoded 132 amino acids. Phylogenetic analysis revealed that the amino acid sequences homology between mink and ferret was 99.2%,90.0% with Ailuropoda melanoleuca and Canis familiaris. Prokaryotic expression vector pProEX-HTb-IL-4 was constructed and induced by IPTG. The recombinant protein of 15 ku was isolated by SDS-PAGE and detected by Western blotting. Highly purified recombinant protein of mink IL-4 was obtained by His-Trap HP affinity columns method. This research laid the foundation for the further studies on the biological function of mink IL-4 gene.