凋亡相关蛋白apr.docVIP

凋亡相关蛋白Apr nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 作者：李袁飞 师建国 郭爱林 朱国强 闫庆国 【关键词】nbsp; Apr 　　Cloning, sequencing and preliminary expression of apoptosis related protein 2 　　【Abstract】 AIM: To clone and express the apoptosis related protein 2 (Apr2) from the model of the apoptosis cells of HL60. METHODS: The model of apoptosis cells of HL60 was established, total RNA was isolated from the cells and mRNA was reversely transcribed into cDNA. PCR was used to amplify the apr2 coding region and the PCR product was cloned into PGEMT Easy vector and sequenced. It was then subcloned into expression vector PGEX4T2 and induced with IPTG. RESULTS:nbsp; Apr2 gene was cloned into PGEMT Easy vector and the sequence was confirmed. SDSPAGE 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:nbsp; Apr2 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 Apr2s structure and function. 　　【Keywords】 Apr2 gene; apoptosis; cloning ; fusion protein; inclusion bodies 　　【摘要】 目的： 从HL60细胞凋亡模型中克隆凋亡相关蛋白Apr2编码区基因,并对其进行表达,为进一步研究Apr2的结构功能及多克隆抗体的制备奠定基础.方法： 建立HL60细胞凋亡模型,提取HL60凋亡细胞总RNA,以RTPCR方法获取Apr2 cDNA编码区全序列,将其与PGEMT Easy载体连接,转化E.coli DH5α,构建重组克隆载体PGEMTEasy/apr2,测序正确后,将目的片段亚克隆入PGEX4T2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Apr2 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HL60中Apr2 cDNA编码区的克隆及其融合蛋白表达产物. 　　【关键词】 Apr2基因;凋亡;克隆;融合蛋白;包涵体 　　0引言 　　凋亡是一个受遗传调控的自身编码死亡方式，涉及许多基因的共同作用［1］，这些基因中仍有相当一部分未知. 朱峰等［2］人通过建立HL60细胞凋亡模型，运用改良消减杂交法（improved subtractive hybirdization, ISH）克隆出了10个凋亡相关新基因，并在GenBank中登录，其中凋亡相关蛋白Apr2的生化性质、结构、功能以及与凋亡的确切关系还不清楚，国内外文献尚无研究报道. 我们利用HL60细胞凋亡模型，对Apr2基因编码区进行克隆、表达及序列分析