猪ISG15基因的克隆表达与多克隆抗体的制备

Cloning and expression of pig ISG15 gene and preparation of the polyclonal antibodies

本研究从家猪外周血淋巴细胞中提取总RNA,通过RT-PCR克隆出家猪ISG 15基因,经测序和序列分析,将ISG 15片段与p ET28a质粒连接,构建重组原核表达载体p ET28a-ISG15;经IPTG诱导表达后,通过Ni柱纯化,以获得ISG15蛋白。用纯化的ISG15蛋白免疫家兔,制备多克隆抗体。结果显示,家猪ISG 15基因CD S区长504 bp,编码167个氨基酸,包括两个泛素样结构域(N端第3~75位和C端第81~156位氨基酸肽段)及C末端保守序列为LRLRGG;W estern-blot分析表明重组ISG15蛋白的免疫原性良好;经ELISA测定血清中抗体的效价为1∶102 400。上述研究结果为进一步揭示家猪ISG15蛋白的功能奠定了基础。

The ISG15 gene of pig was amplified by RT-PCR from peripheral blood lymphocytes cells. The pig ISG15 gene was cloned into pET28a , expressed with a 6×histidine tag by IPTG and purified by Niv affinity chromatography. Rabbits were immunized with purified ISG15 protein to obtain the polyclonal antibodies .The pig ISG15 gene was found to consist of 504 bp encoding 167 amino acid residues. Compared with ISG15 gene of other species,the most conserved regions of the pig ISGI5 peptide were found to be the tandem ubiquitin-like domains （No. 3-75 amino acids in theN-terminal and No, 81-156amino acids in the C-terminal）and LRLRGG conjugating motif in the C-terminal,characteristic of the ISG15 proteins. Western-blot analysis showed that the antibodies had good specificity. The ELISA results showed that the antibody titer of polyclonal antibodies was 1： 102 400. This study result lays a foundation for further studies of the function of pig ISGIS.