卟啉病相关胆色素原脱氨酶基因的克隆与原核表达

Porphobilinogen deaminase of porphyria cloning and prokaryotic expression

目的主要为了获得胆色素原脱氨酶(PBGD)在大肠杆菌中的原核重组表达蛋白,并对其进行纯化,为下一步研究PBGD结构及功能提供实验基础。方法以人细胞中的总RNA逆转录得到的c DNA为模板,PCR扩增,构建p ET28a(+)-PBGD重组质粒。把含有外源片段的p ET28a-PBGD重组质粒转入大肠杆菌BL21中进行诱导表达,经His纯化方法获得高纯度的PBGD融合蛋白。结果 PCR扩增出大小约1 500 bp的特异PBGD c DNA片段;成功构建了p ET28a(+)-PBGD融合表达载体;该表达载体融合表达的蛋白分子量约为44 k D,且大部分纯化蛋白存在于包涵体中。胆色素原脱氨酶的酶活性检测中发现,PBGD的最适酶反应温度为36℃,最适酶反应p H为7.5。结论该实验已成功地克隆出编码正确的PBGD基因片段,并构建了高效原核融合表达载体:p ET28a(+)-PBGD。获得PBGD在大肠杆菌中表达的重组蛋白,并纯化至均一,为PBGD结构和功能的研究提供基础。

[Objective] Express and purify Prophobilinogen Deaminase （PBGD） in E. coli and provide basis for the study of structure and function of PBGD. [Methods] Human cell RNAs were extracted and reverse transcribed to cDNA. PBGD was amplified and inserted into prokaryotie expression vector pET28a （＋）. The recombinant vector was transformed into BL21. PBGD expression was then induced by IPTG. The expressed His6-tagged protein was purified by Ni2＋ affinity chromatography column and characterized by SDSPAGE and Western blotting. [Results] PBGD 1 500 bp gene was amplified and sequencing result was correct; the pET28a （＋）-PBGD recombinant vector was successfully constructed; SDS-PAGE analysis showed that the fusion protein was about 44 kD, and a small portion of the protein was expressed in the supernatant; the best temperature for PBGD is 36℃, and the optimum pH of PBGD is 7.5. [Conclusion] The PBGD gene was successfully cloned into pET28a （＋） expression vector and purified by Ni2＋ affinity chromatography column.