本文選題：乙型肝炎病毒 + 復(fù)制型載體��； 參考：《第三軍醫(yī)大學(xué)》2013年博士論文

【摘要】：背景和目的： 將病毒載體設(shè)計成能夠攜帶外源基因并按著親代病毒的侵入路徑到達(dá)細(xì)胞體內(nèi)的病毒變異體是病毒載體研究的新思路。對一些病毒家族來說，攜帶報告基因的復(fù)制型載體能夠在簡化和量化檢測復(fù)制和感染、識別抗病毒和病毒易感細(xì)胞等方面成為有力的工具。乙型肝炎病毒(hepatitis B virus, HBV)，是引起乙型病毒性肝炎的小包裹DNA病毒，原則上在這個僅有3.2kb的基因組內(nèi)插入外源基因不可避免地會影響本身的復(fù)制元件。HBV復(fù)制子通過前基因組RNA(pregenomic RNA, pgRNA)逆轉(zhuǎn)錄而來，pgRNA作為雙順反子mRNA，在形成核心蛋白(C)和逆轉(zhuǎn)錄酶(Pol)方面也是必需的， C和Pol開放讀碼框架(open reading frames, ORF)有150bp的重疊區(qū)。Pol ORF的下游區(qū)翻譯一般不涉及內(nèi)部核糖體進(jìn)入位點(internal ribosome entry site, IRES)。我們設(shè)想將C、P重疊區(qū)域拉開，插入兩個IRES分別用于表達(dá)外源基因和P蛋白，產(chǎn)生一個有功能的且不影響包膜蛋白表達(dá)的三順反子pgRNA。為了減少對基因組長度的影響，我們利用僅含22個nt的RNA結(jié)合基序蛋白3(RNA-binding motif protein3, Rbm3) IRES，構(gòu)建了分別攜帶399bp的殺稻瘟菌素抗性基因(blasticidin resistance, BsdR)和720bp的人綠色熒光蛋白(humanized Renilla green fluorescent protein, hrGFP)的HBV載體，研究發(fā)現(xiàn)仍有復(fù)制能力，能產(chǎn)生同野生型HBV一樣的病毒蛋白，所形成的的病毒顆�？捎糜诟腥綡epRG細(xì)胞。這種新型的攜帶外源基因的HBV載體將成為研究HBV的有力工具。 方法：本課題分三部分進(jìn)行探討。 第一部分：22-nt Rbm3IRES在HBV前基因組RNA上的表達(dá)活性研究 1、構(gòu)建攜帶CMV啟動子和增強(qiáng)型綠色熒光蛋白(Enhanced Green FluorescentProtein, EGFP)的四種質(zhì)粒： I： pCH-EMCV IRES-EGFP(包含腦炎心肌炎病毒(Encephalomyocarditis virus, EMCV) IRES及EGFP)；II：pCH-22nt IRES-EGFP(包含Rbm3IRES及EGFP)；III：pCH-BsdR-22nt IRES-EGFP(為三順反子載體，依次包含Rbm3IRES，BsdR，Rbm3IRES及EGFP)；IV：pCH-pATG-EGFP(包含HBV C基因N端部分與EGFP)。ELISA檢測I、II、III載體轉(zhuǎn)染HepG2或Huh7細(xì)胞后72h的上清液中HBeAg的表達(dá)，并觀察四種載體轉(zhuǎn)染后EGFP的表達(dá)。 2、構(gòu)建攜帶HBV C基因啟動子和熒光素酶基因(Renilla luciferase, RLuc)的四種質(zhì)粒：I：pHBV-EMCV IRES-RLuc(包含EMCV IRES及RLuc)；II：pHBV-22ntIRES-RLuc(包含Rbm3IRES及RLuc)；III：pHBV-BsdR-22nt IRES-RLuc(為三順反子載體，依次包含Rbm3IRES，BsdR，Rbm3IRES及RLuc)；IV：pHBV-pATG-RLuc(包含HBV C基因N端部分與RLuc)。四種質(zhì)粒分別同對照質(zhì)粒pGL3-Control共轉(zhuǎn)染HepG2或Huh7細(xì)胞，通過雙熒光素酶檢測試劑盒測定熒光素酶值。 第二部分：復(fù)制型HBV載體的構(gòu)建及其表達(dá)與復(fù)制能力研究 構(gòu)建兩種復(fù)制型HBV載體：pCH-BsdR和pCH-hrGFP。兩個質(zhì)粒和攜帶野生型HBV的質(zhì)粒pCH-3093分別轉(zhuǎn)染肝癌細(xì)胞系HepG2和Huh7細(xì)胞。通過熒光顯微鏡觀察外源基因hrGFP的表達(dá)，Bsd篩選細(xì)胞克隆。Northern blot檢測質(zhì)粒轉(zhuǎn)染細(xì)胞后RNA的表達(dá)。Western blot、Native western blot分別用于檢測HBV包膜、核心蛋白和組裝的核心顆粒。ELISA測定細(xì)胞上清液中HBsAg和HBeAg的水平。內(nèi)源性聚合酶反應(yīng)(endogenous polymerase reaction, EPR)檢測功能性P蛋白表達(dá)。Southern blot檢測HBV復(fù)制中間體。熒光定量PCR法分析細(xì)胞上清液中HBV DNA含量。CsCl密度梯度離心法分離細(xì)胞上清液中的病毒顆粒。 第三部分：復(fù)制型HBV載體的感染特性研究 野生型HBV質(zhì)粒pCH-3093、pCH-BsdR和pCH-hrGFP分別轉(zhuǎn)染HepG2細(xì)胞，通過聚乙二醇8000沉淀上清中的重組病毒顆粒，用于感染HepRG細(xì)胞，感染8天后，經(jīng)Northern blot檢測HBV RNA的水平，ELISA測定細(xì)胞上清液中的HBsAg和HBeAg。病毒顆粒與高效價HBV免疫球蛋白（hepatitis B immuno-globulin, HBIG）預(yù)孵育1h后再感染HepRG細(xì)胞以驗證重組HBV顆粒是否能被抗體阻斷。 結(jié)果： 第一部分：22-nt Rbm3IRES在HBV前基因組RNA上的表達(dá)活性研究1、I、II、III號載體轉(zhuǎn)染HepG2和Huh7后，HBeAg表達(dá)均無顯著差異。從第一組質(zhì)粒轉(zhuǎn)染HepG2后觀察熒光強(qiáng)度分析，攜帶Rbm3IRES雙順反子載體(II)中Rbm3IRES的翻譯起始效率明顯強(qiáng)于EMCV IRES(I)；相對于雙順反子載體(II)，在三順反子載體上(III)串聯(lián)的第二個Rbm3IRES引導(dǎo)的EGFP表達(dá)強(qiáng)度下降較多；但是仍明顯高于HBV自身翻譯起始序列引導(dǎo)的EGFP表達(dá)水平(IV)。2、從第二組質(zhì)粒分別轉(zhuǎn)染HepG2和Huh7細(xì)胞后有相似的熒光素酶結(jié)果，Rbm3IRES產(chǎn)生的熒光素酶活性(II)為EMCV IRES產(chǎn)生熒光素酶活性(I)的兩倍有余（II VS I，P＜0.01）。三順反子載體上Rbm3IRES產(chǎn)生的熒光素酶活性較雙順反子明顯下降(III VSII，P＜0.01)，但是仍明顯高于HBV自身翻譯起始序列產(chǎn)生的熒光素酶活性(III VS IV，P＜0.05)。 第二部分：復(fù)制型HBV載體的構(gòu)建及其表達(dá)與復(fù)制能力研究 成功構(gòu)建HBV載體pCH-BsdR和pCH-hrGFP。轉(zhuǎn)染細(xì)胞后能觀察到高水平綠色熒光蛋白表達(dá)，經(jīng)Bsd篩選可形成穩(wěn)定的Bsd抗性細(xì)胞克隆。均可產(chǎn)生攜帶外源基因的pgRNA，，有同野生型HBV相似的核心和包膜蛋白，載體之間HBsAg和HBeAg的分泌量無明顯差異。BsdR載體轉(zhuǎn)染細(xì)胞后能翻譯出有功能的P蛋白。BsdR載體的復(fù)制能力為野生型HBV的40%，hrGFP載體復(fù)制能力明顯減弱。然而，這兩個載體都能夠形成有包膜的病毒。 第三部分：復(fù)制型HBV載體的感染特性研究 利用pCH-BsdR和pCH-hrGFP能夠制備重組病毒顆粒，能夠感染HepRG細(xì)胞。感染8天后，Northern blot可以檢測到前基因組和亞基因病毒RNA，ELISA測定出同野生型HBV一樣的HBsAg和HBeAg的分泌趨勢，HBV-hrGFP病毒感染HepRG后可以檢測到hrGFP的表達(dá)。通過抗體阻斷實驗證實重組HBV通過與野生型HBV相同的方式(通過HBV外膜蛋白)感染HepaRG細(xì)胞。 結(jié)論： 1、不管是用CMV啟動子還是HBV C基因啟動子驅(qū)動，Rbm3IRES都比EMCVIRES有較高的翻譯起始效率。在HBV C基因和P基因之間插入了外源基因的三順反子載體中，串聯(lián)的兩個Rbm3IRES均有能力引導(dǎo)翻譯起始過程。 2、把C、P蛋白分開表達(dá)，利用2個僅22-nt的強(qiáng)效IRES分別表達(dá)外源基因和P蛋白，保持了各結(jié)構(gòu)蛋白的完整性又盡量少擴(kuò)充基因組容量，實現(xiàn)載體功能并保持復(fù)制能力。 3、利用構(gòu)建的HBV載體制備的重組HBV顆粒感染HepRG細(xì)胞，證明其能夠像野生型HBV那樣具有感染能力。 4、由于大量的報告基因和效應(yīng)基因的長度一般小于500bp，這種新型的HBV載體有望成為研究和戰(zhàn)勝HBV的有力工具。
[Abstract]:Background and purpose:
Virus vectors are designed to be capable of carrying foreign genes and viral variants reaching the cell by the invasion pathway of the parent virus is a new idea for viral vector research. For some virus families, a replicative vector carrying the reporter gene can be used to simplify and quantify the remanufacture and infection, and to identify the virus and virus susceptibility. Hepatitis B virus (HBV), a small package of viral hepatitis B, is a small package DNA virus. In principle, the insertion of foreign genes in this only 3.2kb genome inevitably affects the replicas of the replicas of the replicating element,.HBV, to be reversed through the pre genomic RNA (pregenomic RNA, pgRNA). PgRNA is also necessary for the formation of core protein (C) and reverse transcriptase (Pol), and the C and Pol open code framework (open reading frames, ORF) has a 150bp overlap region for downstream region translation, which is generally not involved in the internal ribosome entry site. The overlap area was opened, and two IRES were inserted to express foreign genes and P proteins to produce a functional and not affect the expression of the envelope protein to reduce the effect on the length of the genome. We used the RNA binding protein 3 (RNA-binding motif protein3, Rbm3) IRES, which contained only 22 NT (RNA-binding motif protein3, Rbm3) IRES, respectively. With 399bp blasticidin resistance (BsdR) and 720bp human green fluorescent protein (humanized Renilla green fluorescent protein, hrGFP) HBV carrier, it is found that it is still replicating, producing virus egg white like the wild type, and the virus particles can be used to infect the cells. A new HBV vector carrying exogenous genes will become a powerful tool for studying HBV.
Methods: this topic is divided into three parts.
Part one: the expression activity of 22-nt Rbm3IRES on HBV genome RNA.
1, four plasmids carrying CMV promoter and Enhanced Green FluorescentProtein (EGFP) were constructed: I:pCH-EMCV IRES-EGFP (including Encephalomyocarditis virus, EMCV) IRES and EGFP). The three CIS trans subvector containing Rbm3IRES, BsdR, Rbm3IRES and EGFP, IV:pCH-pATG-EGFP (including N terminal part of HBV C and EGFP).ELISA detection I, II, and the expression of the expression in the supernatant after transfection of the four vectors and observe the expression after transfection.
2, four plasmids carrying the HBV C gene promoter and the luciferase gene (Renilla luciferase, RLuc) were constructed: I:pHBV-EMCV IRES-RLuc (including EMCV IRES and RLuc); II:pHBV-22ntIRES-RLuc (including Rbm3IRES and luciferase); PHBV-pATG-RLuc (including the N terminal part of the HBV C gene and RLuc). The four plasmids co transfected HepG2 or Huh7 cells with the control plasmid pGL3-Control, and the luciferase value was measured by the double luciferase detection kit.
The second part: Construction of replication HBV vector and its expression and replication ability.
Two kinds of replicative HBV vectors were constructed: two plasmids of pCH-BsdR and pCH-hrGFP. and plasmid pCH-3093 carrying wild type HBV transfected to HepG2 and Huh7 cells respectively. The expression of exogenous gene hrGFP was observed by fluorescence microscope, and Bsd screening cell clone.Northern blot detection plasmid transfected cells were expressed. Western blot was used to detect HBV envelope, core protein and core particles of assembly,.ELISA determination of HBsAg and HBeAg in cell supernatant. Endogenous polymerase reaction (endogenous polymerase reaction, EPR) detected functional P protein expression.Southern replication intermediate. Fluorescence quantitative analysis of cell supernatant HBV DNA content and.CsCl density gradient centrifugation were used to isolate virus particles from the supernatant.
The third part: the infectious characteristics of replicating HBV vectors.
The wild type HBV plasmids pCH-3093, pCH-BsdR and pCH-hrGFP were transfected to HepG2 cells respectively. The recombinant virus particles in the supernatant of PEG 8000 were used to infect HepRG cells. After 8 days of infection, the level of HBV RNA was detected by Northern blot, and HBsAg in the cell supernatant and high effective valence immunoglobulin in the cell supernatant were determined. Atitis B immuno-globulin (HBIG) was incubated with 1H to infect HepRG cells to verify whether recombinant HBV particles could be blocked by antibodies.
Result錛

本文編號：1804141