本文關(guān)鍵詞： HCV NS5B蛋白 轉(zhuǎn)基因小鼠模型　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目前全世界有1.7億人感染了丙型肝炎病毒(Hepatitis C virus, HCV),HCV感染慢性化往往可以發(fā)展為嚴(yán)重的肝臟疾病,如脂肪肝、肝硬化和肝癌,嚴(yán)重危害人類(lèi)健康。迄今為止,尚未有疫苗可以預(yù)防HCV感染,而且抗病毒治療成本高、副作用大,療效有限。因此丙型病毒性肝炎是繼乙型病毒性肝炎之后的又一種引起全球廣泛關(guān)注的病毒性肝炎傳染病。 長(zhǎng)期以來(lái),人們一直致力于對(duì)HCV的防治研究,但是研究進(jìn)展緩慢。這主要是因?yàn)镠CV具有較高的變異性,缺乏穩(wěn)定的HCV感染動(dòng)物模型,嚴(yán)重阻礙了對(duì)HCV致病機(jī)制、抗病毒藥物篩選和疫苗研究的進(jìn)程。目前HCV動(dòng)物模型的研究主要集中在感染動(dòng)物模型、人-鼠肝臟嵌合小鼠模型和轉(zhuǎn)基因小鼠模型三個(gè)方面,但都存在很大的局限性。 HCV NS5B是一種RNA依賴(lài)的RNA聚合酶(RdRp),在HCV復(fù)制中起著關(guān)鍵作用。并且鑒于NS5B的晶體結(jié)構(gòu)已經(jīng)明確,對(duì)其功能的研究也較為深入,因此NS5B是目前抗HCV的理想靶標(biāo)之一。本研究通過(guò)聯(lián)合使用Tet-on四環(huán)素調(diào)控系統(tǒng)和Cre/LoxP基因敲除系統(tǒng),建立了一個(gè)可在肝臟組織嚴(yán)格調(diào)控表達(dá)HCV NS5B蛋白的三轉(zhuǎn)基因小鼠模型,研究?jī)?nèi)容和結(jié)果如下; 1.構(gòu)建受Tet-on和Cre/LoxP系統(tǒng)雙重調(diào)控的HCV NS5B真核表達(dá)載體 以真核表達(dá)載體pBI-3為載體構(gòu)建骨架,在其啟動(dòng)子的下游依次插入luc報(bào)告基因、BGH PolyA和ns5b基因片段,并分別在luc報(bào)告基因的上游和BGH PolyA尾的下游各引入一個(gè)LoxP位點(diǎn),成功構(gòu)建了可受Tet-on四環(huán)素調(diào)控系統(tǒng)和Cre/LoxP基因敲除系統(tǒng)雙重調(diào)節(jié)控制的HCV NS5B真核表達(dá)載體,命名為PBI-3/luc-BGH PolyA-NS5B。這種雙調(diào)控設(shè)計(jì)不但可以更為嚴(yán)格的避免漏過(guò)性表達(dá),而且雙報(bào)告基因的設(shè)計(jì)為后期實(shí)驗(yàn)篩選背景表達(dá)低和誘導(dǎo)性強(qiáng)的轉(zhuǎn)基因小鼠提供了簡(jiǎn)便、靈敏、準(zhǔn)確的檢測(cè)方法。PBI-3/luc-BGH PolyA-NS5B真核表達(dá)載體的成功構(gòu)建為可嚴(yán)格調(diào)控HCV NS5B蛋白表達(dá)轉(zhuǎn)基因小鼠的建立奠定了良好的基礎(chǔ)。 2.建立在Dox誘導(dǎo)下可嚴(yán)格調(diào)控表達(dá)NS5B蛋白的三轉(zhuǎn)基因小鼠 將PBI-3/luc-BGH PolyA-NS5B載體線性化后送往上海南方模式生物研究中心制備在Tet-on和Cre/LoxP系統(tǒng)雙重調(diào)控下表達(dá)HCV NS5B蛋白的轉(zhuǎn)基因小鼠,結(jié)果獲得了6只NS5B轉(zhuǎn)基因小鼠首建鼠(F0)。將該首建鼠進(jìn)行PCR及Southern bolt鑒定后與C57BL/6小鼠雜交獲得第一代小鼠(F1), PCR法篩選ns5b基因陽(yáng)性小鼠。經(jīng)篩選獲得的ns5b基因陽(yáng)性的小鼠一部分與C57BL/6小鼠繼續(xù)交配,繁殖擴(kuò)群,用PCR法檢測(cè)其是否能夠?qū)⑼庠椿蚍�(wěn)定遺傳;而另一部分與共表達(dá)rtTA和Cre的雙轉(zhuǎn)基因小鼠(rtTALAP-1/LC-1)雜交,篩選共表達(dá)ns5b、Lap、Cre基因的三轉(zhuǎn)基因小鼠。用PCR法分別檢測(cè)ns5b基因、Lap基因和Cre基因片段,三種片段均陽(yáng)性的三轉(zhuǎn)基因小鼠用Dox(鹽酸強(qiáng)力霉素)飲水飼喂一周,小鼠腹腔注射熒光素,用小動(dòng)物在體光學(xué)成像分析系統(tǒng)下檢測(cè)三轉(zhuǎn)基因小鼠肝臟的發(fā)光信號(hào),用免疫組化法檢測(cè)Cre重組酶和NS5B蛋白在小鼠肝臟組織中的表達(dá)情況。結(jié)果發(fā)現(xiàn)三轉(zhuǎn)基因小鼠在Dox誘導(dǎo)一周后,只在肝臟部位采集到強(qiáng)烈的特異性熒光信號(hào),其他部位未采集到信號(hào),說(shuō)明該三轉(zhuǎn)基因小鼠的調(diào)控性和特異性均良好。免疫組化法證實(shí)Cre重組酶特異性定位于肝細(xì)胞胞核,NS5B蛋白特異性分布于肝細(xì)胞胞漿。 綜上所述,通過(guò)聯(lián)合使用Tet-on四環(huán)素調(diào)控系統(tǒng)和Cre/LoxP基因敲除系統(tǒng)建立了三轉(zhuǎn)基因小鼠模型,其目的基因NS5B表達(dá)的誘導(dǎo)性和肝臟靶向性均良好,為后期評(píng)價(jià)該三轉(zhuǎn)基因小鼠用于HCV RdRp抑制劑篩選的穩(wěn)定性和有效性提供了實(shí)驗(yàn)基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R-332

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本文編號(hào)：1553592