【作者】 王宇；

【导师】 苟克勉；

【作者基本信息】 中国农业大学 ， 生理学， 2004， 硕士

【摘要】 哺乳动物体内能够自身合成饱和脂肪酸和单不饱和脂肪酸，但由于缺乏在第九位碳原子以上位置引入不饱和双键的去饱和酶(脱氢酶)。所以不能自身合成必需脂肪酸，需要从植物及海产品中摄取。本试验试图构建一个肌肉组织特性的ω-6脂肪酸去饱和酶(脱氢酶)真核表达载体，通过原核显微注射法把棉花的ω-6脂肪酸去饱和酶导入到小鼠体内，以小鼠为动物模型，建立动物体内的必需脂肪酸合成代谢通路。同时构建一个ω-6脂肪酸去饱和酶的原核表达载体，通过原核表达来生产ω-6脂肪酸去饱和酶。使用得到的蛋白来生产抗体用于下一步表达检测的Western印迹试验。 本实验以pEGFP-N1质粒为骨架载体，用酶切连接的方法构建一个顺序含有α-actin启动子、FAD2 cDNA、SV40 polyA加尾信号的真核表达载体，双切线性化后回收，使用回收的表达载体经原核显微注射生产转基因小鼠。采用聚合酶链式反应(PCR)和Southern印迹对阳性鼠进行检测。以PCR及酶切连接的方法构建一个含有FAD2 cDNA编码序列的原核表达载体，此蛋白的N端融合有S-tag标签，便于对表达的蛋白进行Southern印迹检测及纯化。 经受精卵原核显微注射后移植到受体母鼠，第一批移植受体后产仔34只，经PCR检测阳性为3只。 原核表达载体pETFAD2转化到表达菌株BL21(DE3)后，经IPTG诱导得到了目标蛋白。更多还原

【Abstract】 BACKGROUND/AIM OF RESEARCHMammals only can pruduce saturated fatty acids and monounsaturated fatty acids. They cannot naturally produce essential fatty acids - beneficial nutrients found mainly in plant and fish oil, so they must rely on a dietary supply. Here we want to establish transgenic mice carry a tissue-specific expression gossypium hirsutum ω -6 fatty acid desaturase(FAD2) gene which can add a double bond into an monosaturated fatty-acid hydrocarbon chain and convert oleic acid to linoleic. Those mice will be a model to establish a pathway of fatty acids metabolism.A prokaryotic expression vector will be established at the same time. On one hand, antibody is necessary to western blot assay. The protein can be used to generate antibody. On the other hand, ω-6 fatty acid desaturase injection assay will be performed to check whether a metabolic pathway is established.METHORDS OF RESEARCHThree plasmids vector with expression elements are used to establish a eucaryotic expression vector by restriction enzyme cutting and ligation. This vector is used to pronucleus microinjection. Founder mice will be examined by polymerase chain reaction(PCR) and southern blot.The pET30 a+ prokatyotic expression vector with a insertion fragment of FAD2 cDNA coding sequence is constructed to express a fusion FAD2 protein with a N-terminal s-tag. With this tag, the protein is easy to detect and purify.RESULTSAfter successfully construction of the eucaryotic expression vector , fertilized eggs were microinjected with linear expression vector and transferred into recipient mice, 34 mice were born and survival. Results of PCR showed that 3 of 34 were positive. Southern hybridization did not show positive. From May the twentith, we begined another microinjection and embryonic transfer and the nearest newborn mice will be cheked at early July.A prokaryotic expression vector was succsessfully constructed and transformed in to E.coli expression strain BL21(DE3). With the IPTG induction, a 50kd protein of ω-6 fatty acid desaturase is expressed.更多还原