摘要
目的 慢性HBV感染是我国的常见病，迄今尚无满意疗法。基因治疗是抗病毒治疗研究的热点，获得高效靶向性导入载体是基因治疗的关键。HBV具有天然嗜肝特性，能在肝细胞内持续复制，反复感染，病毒本身对细胞无明显细胞毒性。HBV具有作为肝靶向性基因治疗载体的基本条件，与其它病毒载体相比，理论上具有明显优势：它可以利用乙肝患者这个天然“包装细胞”，将目的基因的嗜肝性及抗HBV作用在慢性HBV感染者体内长期保持及放大；当HBV复制水平下降时，重组HBV也将随之减少或消失，有可能从根本上克服目前治疗载体的缺陷。本课题对HBV基因组作了三种类型改造：(1)构建C基因截短型HBV载体；(2)构建包膜蛋白突变的HBV载体；(3)构建核心蛋白和包膜蛋白联合突变HBV载体，在细胞和分子水平上观察它们的抗HBV作用，同时探讨adw亚型C基因截短型HBV载体在ayw亚型缺失包装信号辅助质粒辅助下的复制和包装。
方法 1、以野生型HBV质粒adwR9为骨架，构建C基因截短型、野生型C基因真核表达载体pcDNA3-△C(缺失核心蛋白的118-183aa)、pcDNA3-C和C基因截短型HBV载体pHBV-△C(缺失2256-2299nt)。应用脂质体分别将pcDNA3-△C和pcDNA3-C转染HepG2细胞，48小时后提取胞内蛋白，采用Western blot检测蛋白表达。将pcDNA3和pcDNA3-△C分别与adwR9共转染HepG2细胞，采用荧光定量PCR检则培养上清和胞内病毒量，采用ELISA方法检测培养上清中S抗原。将pcDNA3-C分别与pcDNA3-△C和pcDNA3共转染HepG2细胞，采用Native Western blot检测胞内核心颗粒形成。应用脂质体将pHBV-△C单独转染HepG2细胞，以adwR9转染HepG2细胞为对照，采用ELISA方法定量分析胞内和上清中S蛋白表达量和分泌量，采用Western blot检测胞内S蛋白表达。将pHBV-△C与adwR9共转染HepG2细胞，以pcDNA3与adwR9共转染HepG2细胞为对照，采用荧光定量PCR检测培养上清和胞内病毒量。
2、以野生型HBV质粒adwR9为骨架，通过定点突变构建包膜蛋白突变HBV
第三军医大学博士学位论文
载体pHBV一msl(presl区梭基端的1 3aa中，pro和leu密码子突变成^rg)、
pHBV一ms(5 looP56一59aap*LT*15*FN*乏;)和pHBV一mslS。在此基础上，
构建S基因突变型和野生型S基因真核表达载体pcDNA3一ms和pcDNA3一s。
应用脂质体将peDNA3一ms和peDNA3一S分另!转染HepGZ细胞，采用ELIsA
方法检测培养上清和胞内S抗原，采用West。m blot检测胞内蛋白的表达。
将peDNA3一ms和peDNA3分别与adwRg共转染HepGZ细胞，采用ELIsA
方法检测培养上清S抗原，采用荧光定量P〔R检测培养上清和胞内病毒量。
将pHBV一msl、pHBV一ms、pHBV一ms 15和a，lwRg分别转染HepGZ细胞，采
用ELISA方法定量分析胞内S蛋白表达量和上清中S蛋白分泌量，采用荧光
定量PCR检测培养上清和胞内病毒量。应用脂质体将pHBv一ms 15和adwRg
共转染HepGZ细胞，以peDNA3与adwRg岁三转染HepGZ细胞为对照，采用
荧光定量PCR检测培养上清和胞内病毒量。
3、构建核心蛋白和包膜蛋白联合突变HBv载体pHBv一ms 15/△C。应用脂质
体将该载体和pHBv一△C分别与adwRg共转染HePGZ细胞，采用荧光定量
PCR检测培养上清和胞内病毒量。pHBv一△〔与辅助质粒pHBv3 1 42共转染
HepGZ细胞，以pHBV一△C与pGEM共转染HepGZ细胞为对照，采用pCR
检测细胞核内eceDNA和上清中rcnNA形成;采用Native Western blot检测
C蛋白颗粒形成，采用Southern blot检测C蛋白包裹的DNA。
结果1、pcDNA3一C表达的蛋白分子量与野生型adwRg表达的C蛋白分
子量一致，pcDNA3一△C表达的蛋白分子量较野生型adwRg表达的C蛋白分
子量小，表明重组载体能在HepGZ细胞中稳定表达目的蛋白。pcDNA3一△C
与adwRg共转染细胞的上清及胞内病毒量分别为1 .327 x lo4eopieszml和
4.93oXlo4eopies/ml，而peDNA3与adwRg共转染细胞的上清及胞内病毒量
分别为6.13oxlo3eopies/ml和l.s29X108。o:，ies/ml。两组培养上清中s抗原
差异无显著性(P>0.05)。这表明单基因C突变体具有抗HBV作用，但不影
响s蛋白的表达。pcDNA3一C和PcDNA3一△C共转染细胞的胞内核心颗粒条
带较peDNA3一C和peDNA3共转染组条带淡，peDNA3一△C和peDNA3共转
染组无条带，提示突变C蛋白不能形成核心颗粒，但可与野生型C蛋白相互
作用，干扰核心颗粒形成。pHBV一△C转染细胞的胞内S蛋白量与adwRg转
染细胞的基本相同，无显著差异性(P>0.05;pHBv一△C和adwRg共转染细
胞的胞内和上清中病毒量分别为8.977xlo7心opies/ml和9.069X107eopies/ml，
第二军医大学博士学位论文
而adwRg和pcDNA共转染细胞的胞内和[清中病毒量分别为2.379X
logcopies/ml和2.43 x logeopies/ml。这表明HI;ve基因截短对S蛋白的表达
无影响，但可干扰HBV病毒复制。
2、pcDNA3一ms表达的突变S蛋白分子量和野生型S蛋白的分子量一致，约
为27KD，突变体转染细胞胞内和上清中S蛋匀量与野生型adwRg转染细胞
中的基本相同，无显著性差异(P>0.05)。pcDNA3一ms与adwRg共转染细胞
的上清中和胞内病毒量分别为2.43 zx一oZcopi‘:s/ml和9.034火logeopies/ml，
而pcDNA3与adwRg共转染细胞的上清中和胞内病毒量分别为1 .344X
1 o4eop
Objectives Chronic infection with the hepatitis B virus (HBV) is a major problem of public health and a common disease in our country and currently available therapies have limited efficacy. Gene therapy is a hot point in antiviral therapy. It is a key to get a vector targeted the infected tissues or cell in gene therapy. HBV is of hepatotropic specificity. It can specifically infect the liver and continuously replicate in the hepatocytes without obvious cytotoxicity by itself. Hepatitis B virus (HBV; is naturally provided with the prerequisite for being transformed as the liver-targeting gene therapeutic vector. HBV has more distinct advantages than other viral vectors on theory. If it can express anti-HBV products and meanwhile be packaged, the antiviral function will be amplified in vivo. The fact that a large number of HBV-infected humans appear continuously tc harbor the HBV genome and express HBsAg without pathogenic consequences offers a great advantage in terms of long-term maintenance and expression
of a recombinant HBV vector bearing a desired gene. The recombinant HBV genome might be attenuated to the extent that the HBV replication level is reduced. So the limitation of other vectors for HBV gene therapy might be overcome. In this study, the HBV genome was modified as follow: (1) The C gene-truncated HBV vectors were experimentally constructed. (2) The HBV yectors with mutated envelope protein were constructed. (3) The HBV vector with core protein and envelope protein combinablenably mutated was corstructed. These vectors were transfected solely or co-transfected with adwR9 to test its had anti-HBV effect in molecular level. At the same time we had investigated the package of adw subtype C gene-truncated HBV mutant with aid of the ayw subtype helper construct pHBV3142 devoid of the encapsidation signal e.
Methods 1. PcDNA3-AC, pcDNA3-C and pHBV-AC vectors were constructed by molecular cloning and PCR-based deletion from the wild HBV plasmid of adwR9. After transfection with pcDNA3-C and pcDNA3-C by using the
Supported by the National Nature Science Foundation of China, NO: 30371287
liposome method the protein was extracted from the cytoplasm of HepG2 cell line. The expression of mutant protein in the cytoplasmic lysates was detected by Western blot assay. The S protein of HBV was assay by ELISA in the supernatant and viral DNA was detected by Real-time fluorescence quantitative PCR from the supernatant and cytoplasm after co-transfection of pcDNA3-AC and pcDNA3-C with adwR9 respectively into HepG2 cell. The core particles in the transfected cytoplasm were assay by Native Western blot after co-transfection of pcDNA3-AC and pcDNA3 with pcDNA3-C respectively into HepG2 cell. The S protein expression and secret on were assay by ELISA in the supernatant and cytoplasm after transfection w th pHBV-AC into HepG2 cell. The S protein expression was detected by ELISA from the supernatant and the DNA was detected by Real time fluorescence quantitative PCR in the supernatant and cytoplasm after co-transfection with pHBV-AC and adwR9 into HepG2 cell.
2. These envelope protein mutants, pHBV-mSl, pHBV-mS, pHBV-mSIS and pcDNA3-mS, were constructed by molecular cloning and PCR-based mutation from the wild HBV plasmid of adwR9. To observe protein expression and secretion of mutant after transfection with pcDNA3-mS and pcDNA3-S respectively into HepG2 cell the S protein in the supernatant and cytoplasm was assay by ELISA and Western blot was used to detect the protein expression in the cytoplasm. After co-transfection of pcDNA3-mS and pcDNA3 with adwR9 respectively into HepG2 cell the DNA was detected by Real-time fluorescence quantitative PCR in the cytoplasm and supernatant. The S protein was assay by ELISA in the supernatant after co-transfectic n of above both vectors with adwR9. The S protein in the supernatant and cytoplasm was assay by ELISA and the DNA was detected by Real-time fluorescence quantitative PCR in the cytoplasm and supernatant after transfectiou with pHBV-mSl, pHBV-mS, pHBV-mSIS and adwR9 into HepG
引文
1. Ganem D. An advance in liver-specific gene delivery. Proc. Natl. Acad. Sci. USA, 1999, 96: 11696-11697
2. Doronin K, Kuppuswamy M, Toth K, et al. Iissue-specific, tumor-selective, replication- competent adenovirus vector for cancer gene therapy. J virology, 2001, 75:3314-3324
3. Seeger C, Mason W. Hepatitis B virus biology. Microbiol Mol Biol Rew, 2000, 64: 51-68
4. Chaislmchit S, Tyrrell D, Chang Lj. Development of replicative and nonrepli-cative hepatitis B virus vectors. Gene Therapy, 1997, 4:1330-1340.
5. Chiang PW, Hu CP, Su TS, et al. Encapsidation of truncated human hepatitis B virus genomes through trans-complementation of the core protein and polymerase. Virology, 1990, 176:355-361
6. Hanafusa T, Yumoto Y, Hada H, et al. Replication of hepatitis B virus which carries foreign DNA in vitro. Biophys Res Commun, 1999, 262:530-533
7. Protzer U, Nassal M, Chiang PW, et al. Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection. Proc. Natl. Acad. Sci, 1999, 96: 10810-10823.
8．孙殿兴，胡大荣，邬光惠等．基因重组HBV联合表达反义RNA和显性阴性突变体抗HBV作用及HBV包装细胞豸．的构建．中华肝脏病杂志，2002，
10：260-265
9 孙殿兴，胡大荣，邬光惠等．逆转录病毒载体携带HBV载体表达显性阴性突变体抗HBV作用的研究．肝脏，2001，6N S 74
10．孙殿兴，胡大荣，邬光惠等．重组逆转录病毒载体携带HBV载体构建与表达．中华实验和临床病毒学杂志，2002，16：2162-165
11．韩聚强，胡大荣，胡学玲等．以乙型肝炎病毒为载体的基因治疗研究．中华肝脏病杂志，2003，11：344-346
12. Chaislmchit S, Tyrrell DLJ and Chang Lj. Development of replicative and nonreplicative hepatitis B virus vectors. Gene Therapy. 1997, 4:1330-1340
13. Sabine Knig, Gertrud B, Michael N, et al. Mapping of homologous interaction site in the hepatitis B virus core protein. J Vology, 1998, 72(6): 4997-5005.
14. Poisson F, Severac A, Hourioux C, et al. Both pre-S1 and S domains of hepatitis B virus envelope proteins interact with the core particle. Virology, 1997,228: 115-120.
15. Shahabuddin M, Khan AS. Inhibition of human immunodeficiency virus type Ⅰ by packageable, multigenic antisense RNA. Antisense Nucleic Acid Dug Dev, 2000, 10(3): 141-51
16. Chadwick DR, Lever A M. Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type Ⅰ inhibits viral replication at post-transcriptional stages of the life cycle. Gene Ther, 2000, 7(16): 1362-8.
17. Dornburg R, Pomerantz RJ. HIV-Ⅰ gene therapy: promise for the future. Adv Pharmacol, 2000, 49: 229-61.
18. Von Weizsacker F, Kock J, Wieland S, et al. Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis. Hepatology, 1999, 30: 308-315.
19. Koschel M, Oed D, Gerelsaikhan T, et al. Hepatitis B virus core gene mutation which block nucleocapsid envelopment. J Virol, 2000, 74(1): 1-7.
20. Scaglioni P, Melegari M, Takahashi M, et al. Use of dominant negative mutants of the hepadnaviral core protein as antiviral agents. Hepatology, 1996, 24(5): 1010-7
21. von Weizsacker F, Wieland S, Blum HE. Inhibition of viral replication by genetically engineered mutants of the duck hepatitis B virus core protein. Hepatology, 1996, 24(2): 294-9
22．韩聚强，胡大荣，孙殿兴等．携带外源基因的HBV在HepG2细胞中的表达与复制．中华微生物学和免疫学杂志，2003，23(1)：48
23. Gunther S, Fischer L, pult I, et al. Natural y occurring variants of hepatitis B virus. Adv virus Res, 1999, 52: 25-137.
24. Gunther S, Piwon N, Jung A, et al. Erhanced replication contributes to enrichment of hepatitis B virus with a deletion in the core gene. Virology, 2000, 273:286-299
25. Lffler-Mary H, Dumortier J, Klentsch-Zimmer C, et al. Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope
proteins. Virology, 2000, 270:358-367
26. Reinhild P, Rolf E.S. Novel transmembrane topology of the hepatitis B virus envelope proteins. The MBO Journal, 1995, 4 (2): 247-256.
27. Bruss V, Ganem D. The role of envelope protein in hepatitis B virus assembly. Proc Natl Acad Sci U S A, 1991, 88: 1059-1053.
28. Roingeard P, Sureau C, Freschlin M, et al. Immunocytochemical and eletron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells. Hepatology, 1990, 11: 277-85.
29. Huovila AP, Eder AM, Fuller SD. Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol. 1992, 118: 1305-1320.
30．成军，杨守纯主编．现代肝炎病毒分子生，勿学．北京：人民军医出版社，1997：190-195
31. Gunther S, Piwon N, Jung Annegret, et al. Enhanced replication contributes to enrichment of hepatitis B virus with a de ietion in the core gene. Virology, 2000, 273:286-299
32. Kock J, Theilmann L, Galle P, et al. Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus. Hepatology, 1996, 23:405-413
33. Ilan E, Burakova T, Dagan S, et aI. The hepatitis B virus-trimera mouse: a model for human HBV infection and ewluation of anti-HBV therapeutic agents. Hepatology, 1999, 29:553-562.
34. Mazarakis N, Azzouz, M, Rohll, J, et al. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum Mol Genet, 2001, 10:2109-2121
35. Connolly J. Lentiviruses in gene therapy clinical research. Gene Therapy, 2002, 9:1730-1734
36. Seeger C, Mason W. Hepatitis B virus biology. Microbiol Mol Biol Rew, 2000, 64(1): 51-68
37. Junker-Niepmann M, Bartenschiager R, Schaller H. A short cis- acting
sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. J EMBO, 1990, 9(10): 3389-3396
38．韩聚强，胡大荣等．C基因截短HBV载体的复制、包装与表达．中华传染病杂志．2004，已接受．
39. Yoshitaka O, Haruo O, Reiko S, et al. The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Research, 1983, 11(6): 1747-1757