血红素加氧酶1分子克隆及表达载体的构建

Molecular cloning of heme oxygenase-1 and its eukaryon expression vector construction

背景:近年来许多研究发现血红素加氧酶1具有抗炎、抗氧化及抗凋亡等作用,并且在心肌缺血-再灌注损伤等应激反应中有重要的保护作用,从而成为研究热点。目的:克隆大鼠血红素加氧酶1全长基因,并将其于真核表达载体PIRES2-EGFP相连接,构建血红素加氧酶1的真核表达载体。设计、时间及地点:实验于2008-03/06在苏州大学生物技术研究所完成。材料:成年雄性SD大鼠,由苏州大学动物实验中心提供,用于脾脏细胞总RNA的提取;克隆载体PMD18-T购自Takara公司;表达载体PIRES2-EGFP由苏州大学生物技术研究所保存。方法:分离大鼠脾脏获取脾脏细胞,Trizol法提取脾脏细胞的总RNA,经反转录-聚合酶链反应扩增获得血红素加氧酶1基因,将扩增出来的产物与克隆载体PMD18-T连接,转化TOP10感受态细菌,挑取阳性克隆经PCR及酶切鉴定后测序确证。测序正确后抽提质粒作为模板进行PCR,Sal-Ⅰ和BamH-Ⅰ同时双酶切PCR产物和载体PIRES2-EGFP,再在T4DNA连接酶的作用下进行连接,构建其真核表达载体。主要观察指标:血红素加氧酶1基因的克隆和DNA测序结果证实序列是否正确;PIRES2-EGFP/血红素加氧酶1真核表达载体的构建以及测序证实目的基因成功插入载体。结果:①实验完成了大鼠血红素加氧酶1基因的克隆,所获得的序列与GeneBank中收录的大鼠血红素加氧酶1序列完全一致。②构建真核表达载体PIRES2-EGFP/血红素加氧酶1,测序正确说明血红素加氧酶1基因成功插入表达载体PIRES2-EGFP中。结论:实验成功克隆了大鼠血红素加氧酶1基因全长,并构建了其真核表达载体。

BACKGROUND： Recently, many studies have found that heme oxygenase-1 （HO-1） has effects of anti-inflammatory, antioxidation and anti-apoptosis, further more, it plays an important role in stress reaction such as myocardial ischemia-reperfusion injury, which made it become a research focus. OBJECTIVE： To clone the full-length fragment of HO-1 and to connect it with PIRES2-EGFP to construct its eukaryotic expression vector. TIME AND SETTING： The experiment was performed in Institute of Biotechnology, Soochow University from March to June in 2008. MATERIALS： Adult male Sprague Dawley （SD） rats provided by Animal Experimental Center of Soochow University were used for extracting total RNA from spleen cells. PMD18-T cloning vectors were purchased from Takara Company. Expression vectors PIRES2-EGFP were conserved by Institute of Biotechnology, Soochow University. METHODS： Total mRNA was extracted from spleen cells isolated from SD rats by Trizol method. HO-1 genes were cloned through RT-PCR reaction. Their products were connected with clone vectors PMD18-T, and then transformed into the competence bacteria TOP10. The positive clones were chosen to identify by plasmid PCR and restriction enzyme digestion. After the sequence was confirmed, the plasmids were used as templates for PCR. The products and PIRES2-EGFP vectors were cut with Sal-I and BamH-I, and then T4 DNA ligase was adopted to connect and construct their eukaryotic expression vectors. MAIN OUTCOME MEASURES： The clone of HO-1 was observed and the sequence was confirmed by DNA sequencing. The construction of eukaryotic expression vector PIRES2-EGFP/HO-1 was observed and the target gene was inserted into the vector successfully was confirmed by DNA sequencing. RESULTS： （1）The full-length of HO-1 was cloned and its sequence was quite same with the sequence of HO-1 in GeneBank. （2） Eukaryotic expression vector PIRES2-EGFP/HO-1 was constructed, and exactitude gene sequence indicated HO-1 had been inserted into the PIRES2-EGFP vector. CONCLUSION： The full-length of rat HO-1 has been successfully cloned and eukaryon expressing vector PIRES2-EGFP/HO-t was constructed.