凋亡相关蛋白Apr-2的克隆、测序及初步表达

目的: 从HL-60细胞凋亡模型中克隆凋亡相关蛋白Apr-2编码区基因,并对其进行表达,为进一步研究Apr-2的结构功能及多克隆抗体的制备奠定基础.方法: 建立HL-60细胞凋亡模型,提取HL-60凋亡细胞总RNA,以RT-PCR方法获取Apr-2 cDNA编码区全序列,将其与PGEM-T Easy载体连接,转化E.coli DH5α,构建重组克隆载体PGEM-TEasy/apr-2,测序正确后,将目的片段亚克隆入PGEX-4T-2原核表达载体,并转化大肠杆菌, IPTG诱导重组蛋白质表达,分析蛋白质在细菌中的表达分布,进行凝胶自动扫描分析. 结果:序列分析表明,与GenBank中已登录的Apr-2 cDNA编码区序列比较,完全一致.表达的融合蛋白占菌体总蛋白质的40%以上,主要以包涵体的形式存在. 结论:成功的获得了细胞凋亡模型HL-60中Apr-2 cDNA编码区的克隆及其融合蛋白表达产物.

Cloning, sequencing and preliminary expression of apoptosis related protein 2

AIM: To clone and express the apoptosis related protein 2 (Apr 2) from the model of the apoptosis cells of HL 60. METHODS: The model of apoptosis cells of HL 60 was established, total RNA was isolated from the cells and mRNA was reversely transcribed into cDNA. PCR was used to amplify the apr 2 coding region and the PCR product was cloned into PGEM T Easy vector and sequenced. It was then subcloned into expression vector PGEX 4T 2 and induced with IPTG. RESULTS: Apr 2 gene was cloned into PGEM T Easy vector and the sequence was confirmed. SDS PAGE showed that the fusion protein was expressed as inclusion bodies in E. coli . Band density scanning of stained gel was performed to estimate the percentage of the recombinant protein in the total bacteria protein, which was up to 40%. CONCLUSION: Apr 2 gene has been successfully cloned and preliminary expression product of fusion protein has been obtained, which lay the basis for further purification and studies of Apr 2's structure and function.