一种高效hFIX表达载体的构建及其体内外的初步实验研究

发布时间：2017-12-31 13:27

本文关键词：一种高效hFIX表达载体的构建及其体内外的初步实验研究　出处：《扬州大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：以B型血友病为研究对象,构建含有肝脏特异性调控元件的载体表达hFIX基因,利用我们在先前的探索性研究中发现的具有单一位点整合功能的Rep-RBE核心元件建立的位点特异性整合转基因体系,检测评估携有hFIX的载体在模型动物体内的动力学特征,揭示携有hFIX目的基因的质粒的分布、转录、翻译、毒性等一系列的动力学过程和在主要分布器官中的位点特异性整合情况,并根据实验结果在安全性和有效性方面对该系统进行进一步优化和改造。方法：1、构建pRBE HCR hAAT hFIX+pRC、pRBE hAAT hFIX+pRC,细胞水平表达hFIX,并与之前构建的pRBE CMV hFIX+pRC进行比较表达水平的差异。2、在不同种类细胞中(HEK293细胞、A549细胞、HepG2细胞、L02细胞)表达构建质粒,测定hFIX在肝脏特异性调控元件调控下表达差异情况。3、以高压尾静脉的方式注射构建质粒进入小鼠体内,ELISA检测hFIX在体内的表达,PCR检测质粒在体内的分布、转录。4、检测系统安全性,跟踪测定谷丙转氨酶活性变化及肝脏损伤情况。结果：1、构建的pRBE HCR hAAT hFIX+pRC、pRBE hAAT hFIX+pRC在细胞中成功表达,体外实验水平显示hFIX的表达量并无显著性差异。2、通过高压尾静脉注射携带hFIX表达基因的质粒的小鼠体内血浆hFIX水平第一天达到高峰,pRBE HCR hAAT hFIX+pRC组平均浓度达到1534.5±342.8ng/ml,pRBE hAAT hFIX+pRC组27.1±10.2ng/ml,pRBE CMV hFIX+pRC组1338.8±241.0ng/ml,注射后hFIX的量随时间推移而降低,注射后十天pRBE hAAT hFIX基本检测不到表达,pRBE HCR hAAT FIX+pRC和pRBE CMV hFIX+pRC组表达平均水平仍然比较高,且pRBE HCR hAAT FIX+pRC高于pRBE CMV hFIX+pRC组。3、PCR和RT-PCR的结果显示,外源性的DNA在注射后的第一天能够分布到所有实验动物的检查器官(心、肝、脾、肺、肾)并在其中转录。4、尾静脉内快速质粒注射导致的短时间的急性肝损也通过肝脏转氨酶的检测水平和肝脏的组织形态学变化表现出来,这种变化在10天内快速恢复正常。由此证明,该系统可以安全、有效的介导外源基因在动物体内的长期表达。
[Abstract]:Objective: to construct a vector containing liver specific regulatory elements for expression of hFIX gene in type B hemophilia. The site-specific integration transgenic system was established using Rep-RBE core elements with single locus integration found in previous exploratory studies. The kinetic characteristics of the vector carrying hFIX in model animals were evaluated to reveal the distribution, transcription and translation of the plasmid carrying the hFIX target gene. A series of kinetic processes such as toxicity and site-specific integration in the main distributed organs were carried out, and the system was further optimized and modified according to the experimental results in terms of safety and effectiveness. Methods to construct pRBE HCR hAAT hFIX pRBE hAAT hFIX pRC-1, and express hFIX at cell level. The expression level of pRBE CMV hFIX pRC was compared with that of pRBE CMV hFIX pRC. HepG2 cell line (L02 cell) was used to construct the plasmid, and the difference of hFIX expression under the regulation of liver specific regulatory element was measured. 3. The expression of hFIX in mice was detected by Elisa. The distribution of plasmid in vivo, transcription of .4and the safety of the system were detected by Elisa. The changes of alanine aminotransferase activity and liver injury were measured. Results the constructed pRBE HCR hAAT hFIX pRBE hAAT hFIX pRC was successfully expressed in the cells. The experimental level in vitro showed that there was no significant difference in the expression of hFIX. The plasma hFIX level reached a peak on the first day after injection of plasmid carrying hFIX expression gene through high-pressure tail vein in mice. The average concentration of pRBE HCR hAAT hFIX pRC was 1534.5 卤342.8 ng / ml. PRBE hAAT hFIX pRC group (27.1 卤10.2ng / ml), CMV hFIX pRC group (1338.8 卤241.0ng / ml). The amount of hFIX decreased with time after injection, and the expression of pRBE hAAT hFIX was not detected 10 days after injection. The average expression levels of pRBE HCR hAAT FIX pRC and pRBE CMV hFIX pRC were still high. The results of pRBE HCR hAAT FIX pRC were higher than those of pRBE CMV hFIX pRC group. Exogenous DNA can be distributed to all laboratory animal examination organs (heart, liver, spleen, lung, kidney) and transcribed in the first day after injection. The short-term acute liver damage caused by rapid plasmid injection in tail vein was also demonstrated by the level of liver transaminase and the histomorphology of liver. It is proved that the system can safely and effectively mediate the long-term expression of foreign genes in animals.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R450;R3416

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