调控表达HCVNS5B蛋白小鼠模型的建立

发布时间：2018-03-01 21:13

本文关键词： HCV NS5B蛋白转基因小鼠模型　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：目前全世界有1.7亿人感染了丙型肝炎病毒(Hepatitis C virus, HCV),HCV感染慢性化往往可以发展为严重的肝脏疾病,如脂肪肝、肝硬化和肝癌,严重危害人类健康。迄今为止,尚未有疫苗可以预防HCV感染,而且抗病毒治疗成本高、副作用大,疗效有限。因此丙型病毒性肝炎是继乙型病毒性肝炎之后的又一种引起全球广泛关注的病毒性肝炎传染病。长期以来,人们一直致力于对HCV的防治研究,但是研究进展缓慢。这主要是因为HCV具有较高的变异性,缺乏稳定的HCV感染动物模型,严重阻碍了对HCV致病机制、抗病毒药物筛选和疫苗研究的进程。目前HCV动物模型的研究主要集中在感染动物模型、人-鼠肝脏嵌合小鼠模型和转基因小鼠模型三个方面,但都存在很大的局限性。 HCV NS5B是一种RNA依赖的RNA聚合酶(RdRp),在HCV复制中起着关键作用。并且鉴于NS5B的晶体结构已经明确,对其功能的研究也较为深入,因此NS5B是目前抗HCV的理想靶标之一。本研究通过联合使用Tet-on四环素调控系统和Cre/LoxP基因敲除系统,建立了一个可在肝脏组织严格调控表达HCV NS5B蛋白的三转基因小鼠模型,研究内容和结果如下; 1.构建受Tet-on和Cre/LoxP系统双重调控的HCV NS5B真核表达载体以真核表达载体pBI-3为载体构建骨架,在其启动子的下游依次插入luc报告基因、BGH PolyA和ns5b基因片段,并分别在luc报告基因的上游和BGH PolyA尾的下游各引入一个LoxP位点,成功构建了可受Tet-on四环素调控系统和Cre/LoxP基因敲除系统双重调节控制的HCV NS5B真核表达载体,命名为PBI-3/luc-BGH PolyA-NS5B。这种双调控设计不但可以更为严格的避免漏过性表达,而且双报告基因的设计为后期实验筛选背景表达低和诱导性强的转基因小鼠提供了简便、灵敏、准确的检测方法。PBI-3/luc-BGH PolyA-NS5B真核表达载体的成功构建为可严格调控HCV NS5B蛋白表达转基因小鼠的建立奠定了良好的基础。 2.建立在Dox诱导下可严格调控表达NS5B蛋白的三转基因小鼠将PBI-3/luc-BGH PolyA-NS5B载体线性化后送往上海南方模式生物研究中心制备在Tet-on和Cre/LoxP系统双重调控下表达HCV NS5B蛋白的转基因小鼠,结果获得了6只NS5B转基因小鼠首建鼠(F0)。将该首建鼠进行PCR及Southern bolt鉴定后与C57BL/6小鼠杂交获得第一代小鼠(F1), PCR法筛选ns5b基因阳性小鼠。经筛选获得的ns5b基因阳性的小鼠一部分与C57BL/6小鼠继续交配,繁殖扩群,用PCR法检测其是否能够将外源基因稳定遗传;而另一部分与共表达rtTA和Cre的双转基因小鼠(rtTALAP-1/LC-1)杂交,筛选共表达ns5b、Lap、Cre基因的三转基因小鼠。用PCR法分别检测ns5b基因、Lap基因和Cre基因片段,三种片段均阳性的三转基因小鼠用Dox(盐酸强力霉素)饮水饲喂一周,小鼠腹腔注射荧光素,用小动物在体光学成像分析系统下检测三转基因小鼠肝脏的发光信号,用免疫组化法检测Cre重组酶和NS5B蛋白在小鼠肝脏组织中的表达情况。结果发现三转基因小鼠在Dox诱导一周后,只在肝脏部位采集到强烈的特异性荧光信号,其他部位未采集到信号,说明该三转基因小鼠的调控性和特异性均良好。免疫组化法证实Cre重组酶特异性定位于肝细胞胞核,NS5B蛋白特异性分布于肝细胞胞浆。综上所述,通过联合使用Tet-on四环素调控系统和Cre/LoxP基因敲除系统建立了三转基因小鼠模型,其目的基因NS5B表达的诱导性和肝脏靶向性均良好,为后期评价该三转基因小鼠用于HCV RdRp抑制剂筛选的稳定性和有效性提供了实验基础。
[Abstract]:At present, there are 170 million people around the world have been infected with hepatitis C virus (Hepatitis C, virus, HCV, HCV) infection often leads to severe chronic liver disease, fatty liver, liver cirrhosis and liver cancer, serious harm to human health. So far, there has not been a vaccine to prevent HCV infection, and the high cost of antiretroviral treatment, side effects the effect is limited. Therefore, hepatitis C after hepatitis B and a viral hepatitis caused by infectious disease widespread concern around the world.
For a long time, people have been devoted to the study of the prevention and cure of HCV, but the slow progress of the study. This is mainly because of the variability of HCV is high, the lack of a stable animal model of HCV infection, seriously hindered the HCV pathogenesis, screening antiviral drugs and vaccine research process. The present research mainly concentrated in the animal model of HCV infection animal model, three aspects of human liver chimeric mice and transgenic mouse models, but there are many limitations.
HCV NS5B is a RNA dependent RNA polymerase (RdRp), plays a key role in HCV replication. And in view of the crystal structure of NS5B has been clear, the study of its function is more thorough, so NS5B is one of the ideal target for anti HCV at present. This study by using a combination of Tet-on and Cre/LoxP gene tet system knockout system, established a strict regulation of expression in liver tissue of three transgenic mouse model of HCV NS5B protein, research contents and results are as follows;
1. construct a HCV NS5B eukaryotic expression vector controlled by Tet-on and Cre/LoxP systems
To construct eukaryotic expression vector pBI-3 framework as the carrier, in the downstream of the promoter were inserted into Luc reporter gene, BGH PolyA and NS5B Gene Fragments, and were in the Luc gene upstream and downstream the BGH PolyA tail into a LoxP locus, was successfully constructed by Tet-on four ring element control system and Cre/LoxP gene knockout of eukaryotic expression vector of HCV NS5B dual control system named PBI-3/luc-BGH PolyA-NS5B., the double control design can not only more strictly to avoid leakage of expression, and the design of double report gene for later screening experiments and low background expression of transgenic mice induced by strong provides a simple, sensitive and accurate detection methods.PBI-3/luc-BGH PolyA-NS5B eukaryotic expression vector is successfully constructed for the establishment of strict regulation of HCV expression of NS5B protein in transgenic mice and laid a good foundation.
2. the three transgenic mice expressing NS5B protein can be strictly regulated under the induction of Dox
The PBI-3/luc-BGH PolyA-NS5B vector was linearized to transgenic mice Shanghai biomodel Research Center prepared in Tet-on and Cre/LoxP systems under the dual regulation of expression of HCV NS5B protein, we obtained 6 NS5B transgenic mice and founder mice (F0). The rats were first built PCR and Southern bolt identification and C57BL/6 hybrid mice the first generation of mice (F1), PCR method for screening NS5B Gene positive mice were screened. The positive expression of NS5B Gene in mice and C57BL/6 mice to part mating, reproduction of overgroups, using PCR method to detect whether the exogenous gene can be inherited stably; while the other part and the co expression of rtTA and Cre double transgenic mice (rtTALAP-1/LC-1) hybrid screening, co expression of NS5B, Lap, three Cre gene transgenic mice. NS5B Gene were detected by PCR method, Lap gene and Cre gene fragments, three fragments were positive in three transgenic mice Dox (doxycycline hydrochloride) water fed for a week, intraperitoneal injection of fluorescein, with a small animal in vivo optical imaging analysis of light signal detection of three transgenic mice liver system, using immunohistochemical method to detect the recombinant Cre and NS5B protein in liver tissue of mice. The expression of the results showed that three transgenic mice induced by a weeks after Dox, only to specific parts of the liver in the strong fluorescence signal acquisition, other parts were not collected signals that regulation and specificity of the three transgenic mice were good. Immunohistochemical method confirmed that recombinant Cre specific enzyme located in the liver cell nucleus, NS5B protein expressed in liver cells the cytoplasm.
To sum up, through the combined use of Tet-on tetracycline regulatory system and Cre/LoxP gene knockout system established three transgenic mouse model induced liver targeting expression of NS5B gene to HCV RdRp is good for screening inhibitors of stability and validity provides an experimental basis for later evaluation of the three transgenic mice.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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