鸡PERK的原核表达及多克隆抗体的制备

根据鸡的蛋白激酶R样内质网激酶(PERK)基因序列进行抗原肽分析,选择含大片段抗原表位区约1 500 bp的一段基因(15~1 515 bp)设计一对特异性引物后进行RT-PCR扩增,并构建pET-32a(+)-PERK重组质粒,将其转化至BL21(DE3)感受态细胞中.优化诱导表达条件,纯化重组蛋白后免疫兔并制备多克隆抗体.PCR鉴定、酶切鉴定和测序结果表明,pET-32a(+)-PERK重组质粒构建成功.SDS-PAGE电泳鉴定结果表明,重组蛋白在1.0 mmol·L-1IPTG、25℃下诱导9 h时的表达量最大.蛋白纯化结果表明,100 mmol·L-1咪唑洗脱液可较好地洗脱重组蛋白,获得较多的纯化蛋白.免疫结束后,抗体效价检测结果表明,制备的多克隆抗体效价达1∶32 000,可以与重组蛋白特异结合.

Prokaryotic expression and polyclonal antibody preparation of chicken PERK kinase

Based on the antigen peptide sequence of chicken protein kinase R-like ER kinase ( PERK) gene, a ~1500 bp DNA fragment encoding the major epitope domain was amplified by RT-PCR with specific primers, and then was expressed with expression vector pET 32a in BL21 cell ( DE3) . After expression with optimized condition of temperature, time and IPTG concentration, the purified recombinant protein was used to prepare the polyclonal antibody of PERK. Results of PCR, restriction enzyme digestion and sequencing showed that the recombinant plasmid pET-32a (+)-PERK was constructed successfully. SDS-PAGE analysis showed that the optimal expression conditions of recombinant protein were to induce at 25℃ with 1.0 mmol·L-1 IPTG for 9 h. Protein purifica-tion result showed that 100 mmol·L-1 imidazole could elute recombinant proteins effectively, resulting in higher quantity of purified protein. After immunization, the titer of the polyclonal antibody was to 1 : 32000, which could be combined with PERK.