CYP2J基因在双峰驼各组织中的分布与表达及其重组酶体外活性研究

Bactrian camel is a kind of ancient and magical animals, still retaining it's a lot of mystery, and many unknown problems are waiting us there on the camel to further study. The present project is a basic research project which is established on the basis of our previous research works and the unique biological characteristics of the Bactrian camels’ with salt tolerance and do not suffer from salt sensitive hypertension, like to eat some poisonous plants without poisoning. Our research group already accomplished the sequencing and analysis of whole genome of Bactrian Camel, and we found the distribution of camel CYP gene subfamilies are much difference with more CYP2J copies compared with cattle, chicken, horse and human; As well as, we studied the effects of Bactrian camel CYP enzymes on pharmacokinetic characteristics of probe drugs. In present project, firstly we will collect samples from each organ and tissue of Bactrian camel extensively, and extract the total RNA, then to get full CYP2J gene by 3'RACE and 5'RACE. secondly, the in vitro CYP2J expression system will be constructed for obtaining high purity recombinant CYP2J enzyme. Thirdly, the activities of recombinant CYP2J enzyme on metabolism of arachidonic acid, specific drugs and poisons will be studied. As well as, the CYP2J polyclonal antibody will be prepared for studying the expression and distribution of CYP2J protein in tissue of the camel. The achievement of the project will reveal the expression of CYP2J gene subfamily in each organ and tissue of Bactrian camel, and will clarify the significance of CYP2J enzyme on the metabolism of arachidonic acid, specific drugs and poisons in Bactrian camel, and will lay a scientific foundation for the further revealing the Bactrian camel anti salt sensitive hypertension and metabolism of xenobiotics mechanism.

双峰驼仍然保留着它神秘色彩，有很多奥秘等待着我们去研究。在完成双峰驼基因组学研究与双峰驼CYP酶系对探针药物动力学特征的影响等研究基础上，根据双峰驼特有的耐高盐而不患盐敏感性高血压、喜欢采食某些有毒植物而不中毒等生物学特性和CYP2J基因拷贝数明显多于其它物种的特殊性申请本项目。首先广泛采集双峰驼各个器官组织样品，提取总RNA，采用3'RACE和5'RACE获得双峰驼CYP2J全基因；构建CYP2J的体外表达系统，获得高纯度重组表达CYP2J酶，并研究对花生四烯酸、特定药物和毒物的代谢活性；同时，制备CYP2J多克隆抗体，采用Western blot研究CYP2J蛋白在骆驼各组织中的表达与分布。项目成果将揭示双峰驼CYP2J在体内各器官组织中的表达与分布，阐明CYP2J酶对双峰驼花生四烯酸、特定药物和毒物代谢的意义，为进一步揭示双峰驼抗盐敏感性高血压和对外来物质的代谢机理奠定基础。

双峰驼具有特殊的生物学特性和独持的物质代谢能力，且这些特点与其所特有的CYP酶系有着密切关系。在完成双峰驼全基因组学研究与双峰驼CYP酶系对探针药物动力学特征的影响等研究基础上，我们进一步对其CYP2J酶进行深入研究。首先广泛采集双峰驼各个器官组织样品，提取总RNA，采用3'RACE和5'RACE获得双峰驼CYP2J全基因，c DNA全长1770 bp，含有8 bp的5'非翻译区（5'-UTR）和253 bp的3'非翻译区（3'-UTR）及1509 bp 的完整开发阅读框共编码502 个氨基酸残基。生物信息学分析表明其编码的蛋白质预测分子量为57.98 kD、等电点值为8.45、分子式为C2645H4099N713O726S15。且该蛋白为稳定的亲水性蛋白质，不具有信号肽，但有一个跨膜区域（12-35aa），主要锚定于内质网，二级结构主要由α螺旋和无规则卷曲组成。原核表达结果表明，0.5 m M IPTG在大肠杆菌BL21中37℃诱导6 h后，可实现CYP2J蛋白高效表达，且所得纯化蛋白对花生四烯酸和特定药物有一定代谢活性。同时，制备CYP2J多克隆抗体，采用SYBR Green I实时荧光定量PCR法和Western blot法研究CYP2J从基因和蛋白水平上在双峰驼肝脏、心脏、脾脏、肺脏、肾脏、血管、胰腺、十二指肠、空肠、回肠、盲肠、结肠和胃等13种组织中的表达与分布情况。在基因水平上，CYP2J基因在双峰驼胰腺组织中的表达量最高；在蛋白水平上，CYP2J蛋白质在双峰驼肝脏组织的表达量最高。在实验过程中制备的兔抗CYP2J抗体效价高（1:50000）、特异性强和稳定性好，能够满足Western blot 检测要求。实验同时与蒙古牛的相应组织进行了对比研究，CYP2J基因在双峰驼肝脏、胰腺、结肠和十二指肠中的相对表达量均显著高于蒙古牛消化系统对应部位中的表达量。项目成果揭示了双峰驼CYP2J酶对花生四烯酸及特定药物代谢的意义，也阐明了CYP2J在双峰驼体内各器官组织中的表达与分布情况，并为进一步揭示双峰驼抗盐敏感性高血压和对外来物质的代谢机理奠定基础。