【作者】 谢飞；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 “睡美人”(Sleeping Beauty , SB)转座系统属于Tc1/mariner转座子家族,已失活了一千多万年。1997年Ivics等根据积累的系统发生数据,利用生物信息学的方法进行分子重建,终于使其恢复转座活性。近年来对“睡美人”转座系统转座效率和转座机理的研究表明SB转座子系统在转基因,基因筛选,促进外源基因表达及基因治疗等领域具有广阔的应用前景。本研究利用“睡美人”转座子系统,比较睾丸直接注射法和SB系统介导下睾丸注射法转基因小鼠的制备效率,并分析了SB系统中的转座酶基因对基因免疫的增强作用。首先从本实验室已构建的精囊腺组织特异性表达载体(PC-EGFP)切出8.4kb含有EGFP的cDNA及其5’和3’调控序列表达片段,插入到“睡美人”转座子载体PT2-HB中获得PT2-EGFP。随后对睾丸直接注射制备转基因小鼠方法进行初步优化,利用已构建的SB系统和经过双酶切PC-EGFP获得的8.4kbDNA片段分别进行睾丸注射制备转基因动物,利用PCR方法比较转基因动物的制备效率,并对“睡美人”转座子与转座酶的比例进行了初步的摸索。研究结果表明,用4号针头,进行3次注射的公鼠与野生型ICR母鼠合笼后代的产子数及转基因阳性率较理想;经PCR并结合精囊腺和阴道栓的表达检测,SB系统介导的转基因阳性率(43%左右)显著高于普通8.4kb DNA片段的注射组(20%左右),“睡美人”转座子与转座酶的比例在5:1-20:1范围,后代的阳性率差异不显著。本试验同时构建了表达人乳铁蛋白(human lactoferrin,hLF)的真核表达载体pA-hLF,在进行家兔人乳铁蛋白多抗的制备过程中,按照“睡美人”转座酶:PA-LF=4:1的比例用PEI包裹后注射新西兰家兔,并设置PA-LF与PEI包裹,裸露的PA-LF两个注射组与对照组,以评价“睡美人”转座酶对于外源基因表达的促进作用。结果表明:“睡美人”转座酶与PA-LF注射组其抗体效价可达1:6400,高于PEI包裹PA-LF组(1:1600)和裸露质粒注射组(1:2000)。研究结果表明“睡美人”转座酶对基因免疫具有促进作用,可作为一种新型基因佐剂在抗体制备过程中发挥一定的作用。更多还原

【Abstract】 The Sleeping Beauty (SB) transposon is a member of Tc1/mariner family transposons that has been transpositionally inactive for over 10 million years. The SB transposon system was awakened from inactive Tc1-like transposable elements by using molecular phylogenetic data in 1997. Recent studies on its transposition efficiency and mechanism have shown its broad applications in vertebrate animals for gene-screening, gene transfer, and human gene therapy.In this study, the trangenic efficiency introduced by SB system in mouse and effect on gene immunization of Sleeping Transposase was analysed. Firstly, method of injection was optimized by exlporing the size of injector pinhead, injecting times and hormone in this research. The vector PT2-EGFP was constructed by inserting 8.4kb fragment including EGFP and its 5’and 3’regulated sequence from constructed seminal vesicle vector(PC-EGFP) into PT2-HB. Transgenic animals were obtained by direct testis injection and injection mediated by SB transposon system respectively.The transgenic rate were evaluated and compared between these two methods by detection of the fluorescence of vaginal plug and section of seminal vesicle under fluorescence microscope and PCR. In addtion, the different ratio of tansposon and tansposase in SB transposon system was assessed for high transgenic rate. The results demonstrated that the transgenic rate and litter size of offspring in the groups injected by NO.4 pinhead and injected three times were higher. The transgenic rate mediated by SB(43%) was higher than the transgenic rate by direct testisinjection(20%). However, the transgenic rate was not significant different among the groups with different ratio of transposon and transposase from 5:1-20:1. The results demontrated that the Sleeping Beauty system can enhance the transgenic rate.To investigate the effect of Sleeping Beauty transposase on gene immunazition, the vector pA-hLF was constructed and expressed lactoferritin for gene immunization in Newsland rabbits. 8 weeks old rabbits were divided into four groups: group 1, nake vector-pA-hLF;group 2, PEI-coated pA-hLF;group 3, PEI coated complex with Sleeping Transposase and pA-hLF at a molar ratio of 4:1,;group 4, the control group withough injection.Then intrmuscula immunizations were conducted every two weeks in first 6 week and the final injection was given in 18th week. the blood was sampled and evaluated the titer. After the fourth immunizations the third group produce the antibody against hLF by 1:6400 higher than the first group (1:1600) and the second group (1:2000), and the control group showed no antibody. The result indicate that SB tansposase can promote the expression of exogenous gene, which could be used as a new adjuvant in gene immunization.更多还原