关键词: NADP依赖性视黄醇脱氢/还原酶, 选择性剪接, 宫颈癌, 重组表达

Abstract: BACKGROUND AND AIM: To express NRDRB1 in prokaryotic expression system for detecting enzyme activity and preparing polyclonal antibody. MATERIALS AND METHODS: We investigated the mRNA expression of NRDRB1 in cervical squamous carcinoma using RT_PCR. The total NRDR and NRDRB1 sequences were cloned and the coding regions were constructed to the Gateway_based expression vector (pDEST 17), which was transformed into the Escherichia coli(BL21_AI)for protein expression. The recombinant proteins were purified by affinity chromatography. RESULTS: We identified a novel alternatively spliced variant, NRDRB1, in HeLa cell and human cervical squamous carcinoma cells, characterized by a complete deletion of exon 3. The expression vectors of NRDR and NRDRB1 were constructed. The proteins, harvested at the optimal time point (4 hours after induction), were successfully expressed with a 30－50％ expression leve1. The homogeneous proteins were obtained by a one_step affinity chromatography. CONCLUSION: Recombinant NRDR and NRDRB1 from human cervical squamous carcinoma cells were expressed effectively in BL21_AI by pDEST 17.

Key words: NRDR, alternative splicing, cervical carcinoma, recombinant expression