高原鼠兔低氧诱导因子-1α蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of plateau pika HIF-1α protein

高原鼠兔生活在青藏高原海拔3000～5000m的区域,具有极强的低温、低氧耐受能力。低氧诱导因子-1(HIF-1)是一种由HIF-1α和HIF-1β组成的异源二聚体转录因子,在生物体的低氧适应性调节中起着关键作用。低氧主要通过调节HIF-1α蛋白水平来影响HIF-1α的转录活性。本研究用DNA重组技术将高原鼠兔HIF-1α(pHIF-1α)(538/822)编码基因序列亚克隆至原核表达载体PGEX-4T-1中,并在大肠杆菌BL21菌株中进行高效诱导表达,获得可溶性表达产物。将纯化后的GST-pHIF-1α(538/822)融合蛋白作为免疫原免疫家兔制备多克隆抗体。免疫印迹结果表明,制备的抗体能够识别高原鼠兔HIF-1α蛋白。进一步利用亲和纯化的方法对此多克隆抗体进行了纯化。免疫印迹和免疫染色的结果表明,纯化的抗体可以用于检测外源以及内源的高原鼠兔HIF-1α蛋白的表达和定位,为后续的研究提供了重要的实验材料。

The plateau pika （ Ochotona curzoniae） is a high hypoxia-tolerant and cold adapted species living only at 3 000 - 5 000 m above sea level on the Qinghai-Tibetan Plateau. Hypoxia inducible factor-1 （ HIF-1 ）, which is composed of HIF-1α and HIF-1β subunits, is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. HIF-1 transcription activity is mainly regulated through oxygen-dependent modification of HIF-1α protein. The gene sequence encoding pika HIF-1α （pHIF-1α） （538/822） was inserted into PGEX- 4T-1 bacterial expression vector using recombinant DNA techniques. After transformation by the expression vector, E. coli BL21 was induced to highly express the target protein fused by GST protein. After identification and purification, the solubly expressed GST-pHIF-1α （538/822） fusion protein was used to immunize rabbits for preparing polyclonal antibody. The quality of the antibody was identified by Western blot. The results indicated that the polyclonal antibody recognizes plateau pika HIF-1α protein. The polyclonal an- tibody was further affinity purified. Western blot and immunostaining analysis reveal that the purified antibody can detect in vitro and in vivo expression and localization of plateau pika HIF-1α protein and be used in the further study.