携带外源基因的复制型HBV载体的构建

发布时间：2018-04-26 02:41

  本文选题：乙型肝炎病毒 + 复制型载体　； 参考：《第三军医大学》2013年博士论文

【摘要】：背景和目的： 将病毒载体设计成能够携带外源基因并按着亲代病毒的侵入路径到达细胞体内的病毒变异体是病毒载体研究的新思路。对一些病毒家族来说，携带报告基因的复制型载体能够在简化和量化检测复制和感染、识别抗病毒和病毒易感细胞等方面成为有力的工具。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，原则上在这个仅有3.2kb的基因组内插入外源基因不可避免地会影响本身的复制元件。HBV复制子通过前基因组RNA(pregenomic RNA, pgRNA)逆转录而来，pgRNA作为双顺反子mRNA，在形成核心蛋白(C)和逆转录酶(Pol)方面也是必需的， C和Pol开放读码框架(open reading frames, ORF)有150bp的重叠区。Pol ORF的下游区翻译一般不涉及内部核糖体进入位点(internal ribosome entry site, IRES)。我们设想将C、P重叠区域拉开，插入两个IRES分别用于表达外源基因和P蛋白，产生一个有功能的且不影响包膜蛋白表达的三顺反子pgRNA。为了减少对基因组长度的影响，我们利用仅含22个nt的RNA结合基序蛋白3(RNA-binding motif protein3, Rbm3) IRES，构建了分别携带399bp的杀稻瘟菌素抗性基因(blasticidin resistance, BsdR)和720bp的人绿色荧光蛋白(humanized Renilla green fluorescent protein, hrGFP)的HBV载体，研究发现仍有复制能力，能产生同野生型HBV一样的病毒蛋白，所形成的的病毒颗粒可用于感染HepRG细胞。这种新型的携带外源基因的HBV载体将成为研究HBV的有力工具。 方法：本课题分三部分进行探讨。 第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究 1、构建携带CMV启动子和增强型绿色荧光蛋白(Enhanced Green FluorescentProtein, EGFP)的四种质粒： I： pCH-EMCV IRES-EGFP(包含脑炎心肌炎病毒(Encephalomyocarditis virus, EMCV) IRES及EGFP)；II：pCH-22nt IRES-EGFP(包含Rbm3IRES及EGFP)；III：pCH-BsdR-22nt IRES-EGFP(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及EGFP)；IV：pCH-pATG-EGFP(包含HBV C基因N端部分与EGFP)。ELISA检测I、II、III载体转染HepG2或Huh7细胞后72h的上清液中HBeAg的表达，并观察四种载体转染后EGFP的表达。 2、构建携带HBV C基因启动子和荧光素酶基因(Renilla luciferase, RLuc)的四种质粒：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(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及RLuc)；IV：pHBV-pATG-RLuc(包含HBV C基因N端部分与RLuc)。四种质粒分别同对照质粒pGL3-Control共转染HepG2或Huh7细胞，通过双荧光素酶检测试剂盒测定荧光素酶值。 第二部分：复制型HBV载体的构建及其表达与复制能力研究 构建两种复制型HBV载体：pCH-BsdR和pCH-hrGFP。两个质粒和携带野生型HBV的质粒pCH-3093分别转染肝癌细胞系HepG2和Huh7细胞。通过荧光显微镜观察外源基因hrGFP的表达，Bsd筛选细胞克隆。Northern blot检测质粒转染细胞后RNA的表达。Western blot、Native western blot分别用于检测HBV包膜、核心蛋白和组装的核心颗粒。ELISA测定细胞上清液中HBsAg和HBeAg的水平。内源性聚合酶反应(endogenous polymerase reaction, EPR)检测功能性P蛋白表达。Southern blot检测HBV复制中间体。荧光定量PCR法分析细胞上清液中HBV DNA含量。CsCl密度梯度离心法分离细胞上清液中的病毒颗粒。 第三部分：复制型HBV载体的感染特性研究 野生型HBV质粒pCH-3093、pCH-BsdR和pCH-hrGFP分别转染HepG2细胞，通过聚乙二醇8000沉淀上清中的重组病毒颗粒，用于感染HepRG细胞，感染8天后，经Northern blot检测HBV RNA的水平，ELISA测定细胞上清液中的HBsAg和HBeAg。病毒颗粒与高效价HBV免疫球蛋白（hepatitis B immuno-globulin, HBIG）预孵育1h后再感染HepRG细胞以验证重组HBV颗粒是否能被抗体阻断。 结果： 第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究1、I、II、III号载体转染HepG2和Huh7后，HBeAg表达均无显著差异。从第一组质粒转染HepG2后观察荧光强度分析，携带Rbm3IRES双顺反子载体(II)中Rbm3IRES的翻译起始效率明显强于EMCV IRES(I)；相对于双顺反子载体(II)，在三顺反子载体上(III)串联的第二个Rbm3IRES引导的EGFP表达强度下降较多；但是仍明显高于HBV自身翻译起始序列引导的EGFP表达水平(IV)。2、从第二组质粒分别转染HepG2和Huh7细胞后有相似的荧光素酶结果，Rbm3IRES产生的荧光素酶活性(II)为EMCV IRES产生荧光素酶活性(I)的两倍有余（II VS I，P＜0.01）。三顺反子载体上Rbm3IRES产生的荧光素酶活性较双顺反子明显下降(III VSII，P＜0.01)，但是仍明显高于HBV自身翻译起始序列产生的荧光素酶活性(III VS IV，P＜0.05)。 第二部分：复制型HBV载体的构建及其表达与复制能力研究 成功构建HBV载体pCH-BsdR和pCH-hrGFP。转染细胞后能观察到高水平绿色荧光蛋白表达，经Bsd筛选可形成稳定的Bsd抗性细胞克隆。均可产生携带外源基因的pgRNA，，有同野生型HBV相似的核心和包膜蛋白，载体之间HBsAg和HBeAg的分泌量无明显差异。BsdR载体转染细胞后能翻译出有功能的P蛋白。BsdR载体的复制能力为野生型HBV的40%，hrGFP载体复制能力明显减弱。然而，这两个载体都能够形成有包膜的病毒。 第三部分：复制型HBV载体的感染特性研究 利用pCH-BsdR和pCH-hrGFP能够制备重组病毒颗粒，能够感染HepRG细胞。感染8天后，Northern blot可以检测到前基因组和亚基因病毒RNA，ELISA测定出同野生型HBV一样的HBsAg和HBeAg的分泌趋势，HBV-hrGFP病毒感染HepRG后可以检测到hrGFP的表达。通过抗体阻断实验证实重组HBV通过与野生型HBV相同的方式(通过HBV外膜蛋白)感染HepaRG细胞。 结论： 1、不管是用CMV启动子还是HBV C基因启动子驱动，Rbm3IRES都比EMCVIRES有较高的翻译起始效率。在HBV C基因和P基因之间插入了外源基因的三顺反子载体中，串联的两个Rbm3IRES均有能力引导翻译起始过程。 2、把C、P蛋白分开表达，利用2个仅22-nt的强效IRES分别表达外源基因和P蛋白，保持了各结构蛋白的完整性又尽量少扩充基因组容量，实现载体功能并保持复制能力。 3、利用构建的HBV载体制备的重组HBV颗粒感染HepRG细胞，证明其能够像野生型HBV那样具有感染能力。 4、由于大量的报告基因和效应基因的长度一般小于500bp，这种新型的HBV载体有望成为研究和战胜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锛

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