伪狂犬病病毒UL24基因的原核表达及抗UL24蛋白抗体的制备

Prokaryotic expression and antibody preparation for the Pseudorabies virus UL24 protein

为了给伪狂犬病病毒(PrV)UL24蛋白的细胞定位和功能研究提供参考依据,据GenBank公布的PrVUL24基因序列(登录号NC006151)设计1对引物,以质粒pUL24-GFP为模板,用PCR方法扩增出部分缺失的基因片段mUL24(modified UL24)。将mUL24片段定向克隆到原核表达载体pET-32a(+)中,构建融合表达载体pET-UL24。阳性质粒转化宿主菌E.coliBL21(DE3),经IPTG诱导,重组蛋白(His)6-UL24以包涵体的形式获得表达。用纯化后的pET-UL24融合蛋白免疫家兔,ELISA分析表明,在血清效价达到1∶2 560以上,Western-blot分析制备的抗体可以和pET-UL24表达产物发生反应,具有良好的免疫特异性。

From recombinant plasmid pUL24 GFP, the Prv UL24 gene was amplified with a pair of specific designed primers according to the relevant nucleotide sequence on GenBank （NC006151）. The rnUL24 gene with the clustered rare conclous for E. coli at the 3＇ and 5＇ end was directly cloned into the PEF-32a（＋） to get a recombinant plasmid pET-UL24. The pET-UL24 plasmid was transformed into BL21 （DE3） cells and the recombinant protein （His）6-UL24 was expressed in the form of infusion body when in duced with IPTG. Target protein was purified with the Ni^2＋-NTA His · Bind Purification Kit. The purified protein was used to immunize rabbits and the serum was collected when the serum antibody titer reached 1 ： 2 560.