表达HBV preS2S基因重组MVA假病毒颗粒的构建及抗原性鉴定

目的构建携带HBV preS2S基因的重组MVA假病毒颗粒,评价其抗原性。方法将HBV preS2S基因通过基因重组构建入穿梭载体pSC11,得到质粒pSC11-preS2S,将此质粒转染MVA病毒通过同源重组得到MVA—preS2S,通过免疫印迹法鉴定其抗原性。结果利用基因测序,PCR鉴定证实所得假病毒颗粒可以表达HBV preS2S基因,并且具有良好的抗原性,经过9次传代得到单克隆重组病毒。结论本试验成功地构建了表达HBV preS2S基因的重组MVA假病毒颗粒MVA-preS2S,为基因治疗HBV慢性感染做了一项实验性奠基工作。     

幽门螺杆菌glmM DNA重组质粒的构建及体外表达

目的构建幽门螺杆菌(Hp)pVAX1-glmMDNA重组质粒,体外转染SGC-7901细胞并鉴定其表达蛋白的抗原性。方法RT-PCR方法从国际标准株NCTC11637中获取glmM全长基因,克隆插入T载体。测序后经酶切、连接反应将glmM的开放读码框架定向克隆入真核表达载体pVAX1,酶切初步鉴定后测序确认。通过脂质体法将pVAX1-glmMDNA重组质粒转染SGC-7901细胞,检测其转染效率,确定转染成功后RT-PCR法检测glmM在转录水平的表达,ELISA法鉴定表达蛋白的抗原性。结果DNA测序结果显示扩增出的glmM全长基因与GenBank公布的HpglmM序列有96%的同源性。PCR、酶切和测序结果证实glmM目的基因成功克隆入真核表达载体pVAX1。重组质粒转染的细胞经RT-PCR扩增获得1.4kb片段,与目的基因大小相符。ELISA结果显示重组质粒转染细胞的超声破碎物及培养上清液中抗原的滴度比对照组高2倍以上。结论成功构建了pVAX1-glmMDNA重组质粒,其表达蛋白具有良好的抗原性,为进一步研究其抗Hp感染的效果及制备相应有效DNA疫苗奠定基础。     

乙型肝炎病毒全S蛋白与纤维蛋白原α链的相互作用

目的:筛选人肝细胞cDNA文库中与乙型肝炎病毒(HBV)全S蛋白相互作用蛋白的基因,并反向验证HBV全S蛋白候选结合蛋白之间相互作用.方法:将全S基因定向克隆到酵母表达栽体pDEST 32,构建正向筛选的诱饵质粒并Western b1ot法验证其在酵母中的表达.将诱饵质粒与人肝细胞cDNA文库质粒共同转化MaV203酵母细胞,在人肝细胞cDNA文库筛选候选结合蛋白,提取阳性茵落质粒测序.并分析其生物学性质.将筛选出的纤维蛋白原α链中下游序列及不同全S变异株基因,分别定向克隆到pDEST32及pDEST22载体中,利用Western blot法验证表达.将诱饵质粒与猎物质粒共同转化MaV203酵母细胞,以反向酵母双杂交方法验证初筛结果的可靠性及正确性.结果:正向的酵母双杂交实验,经初筛和再转染实验纤维蛋白原α链可与HBV全S蛋白发生相互作用.再以纤维蛋白原α链为靶基因设计诱饵质粒,以四种变异的HBV全S蛋白为靶基因设计猎物质粒,反向酵母双杂交法证实维蛋白原α链中下游可与不同全S变异体(总差异率2%)发生相互作用,纤维蛋白原α链与全S蛋白的结合域可能为病毒蛋白的前268aa.结论:纤维蛋白原β链中下游可与HBV全S蛋白产生特异性结合,其结合域可能与病毒蛋白的前268aa产生相互作用.     

23株HBeAg阳性慢性乙型肝炎患者病毒的全基因组分析

目的研究HBeAg阳性慢性乙型肝炎患者HBV变异特点。方法PCR扩增并克隆HBeAg阳性慢性乙型肝炎患者血清中HBV全基因组DNA,测序并进行基因结构分析。结果获得23株HBV全基因组DNA,它们均属于c或B基因型。与中国HBVB、C基因型参照序列相比,HBeAg阳性慢性乙型肝炎患者来源的HBV在表面抗原、P蛋白、X蛋白的反式激活区及增强子II／核心启动子区发生了一些有意义的共有变异。结论HBV变异可能与HBeAg阳性慢性乙型肝炎的发生、发展有关。     

突变乙型肝炎病毒核心蛋白的克隆及表达

目的 克隆并表达发生长片段缺失的HBV核心蛋白(HBV-C)基因,并对其进行DNA序列、蛋白质结构和抗原性分析.方法 采用PCR方法从1株野生型HBV基因组中扩增得到发生长片段缺失的HBV-C基因,克隆至pUCm-T质粒,进行测序、同源性比较和蛋白质结构分析;再将基因编码区克隆至原核表达载体pET-28a,构建含HBV-C基因的重组表达质粒,转化至大肠埃希菌BL21中进行诱导表达并检测其抗原性.结果 PCR扩增出的HBV-C基因长度经序列分析表明,其核苷酸序列缺失了220 bp至317 bp之间的98个碱基,造成从第74个氨基酸起发生移码突变并失去了抗原性.结论 成功克隆和表达了发生长片段缺失的HBV-C基因.     

应用抑制性消减杂交技术克隆乙型肝炎病毒全S蛋白反式激活基因

目的应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)全S蛋白反式激活基因差异表达的cDNA消减文库,克隆HBV全S蛋白反式激活相关基因.方法以HBV全S表达质粒pcDNA3.1(-)-全S转染HepG2细胞,以空载体pcDNA3.1(-)为对照;制备转染后的细胞裂解液,从中提取mRNA并逆转录为cDNA,经RsaI酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性PCR,将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析.结果成功构建人HBV全S蛋白反式激活基因差异表达的cD-NA消减文库.文库扩增后得到86个白色克隆,进行菌落PCR分析,均得到100-1000 bp插入片段.挑取35个含有插入片段的阳性克隆测序分析,获得33个已知基因序列,和2个未知基因,通过生物信息学分析获得其全长序列,其中之一命名为全S蛋白反式激活基因1(CSTP1),已在GenBank中注册,注册号:AY553877.未知基因的功能还正在研究中.结论应用SSH技术成功构建了HBV全S反式激活基因差异表达的cDNA消减文库.该文库的建立为进一步阐明HBV全S反式调节的靶基因及致肝病发生的分子生物学机制提供理论依据.     

乙型肝炎病毒前S2基因酵母表达载体的构建及表达

目的：探讨乙型肝炎病毒（HBV）前S2蛋白的功能。方法：以质粒pCP10 (含有HBV ayw亚型全长序列)为模板，多聚酶链反应（PCR）扩增HBV前S2基因，克隆到pGEM-T载休整 ，测序鉴定、酶切后回收，与酵母表达质粒pGBKT7连接。将重组载体转化酵母细胞AH109，提取酵母蛋白质，进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹分析。结果：成功构建HBV前S2基因酵母表达载体，Western免疫印迹显示HBV前S2蛋白在酵母细胞中表达，表达产物在胞内存在，分子量24kD左右。结论：HBV前S2蛋白在酵母细胞中表达成功。     

乙型肝炎病毒核壳蛋白变异株在HepG2细胞的HLA-Ⅰ表达

目的:研究HBV adr亚型野生株和核壳蛋白变异株在HepG2细胞表面的HLA-Ⅰ/抗原肽复合物的表达.方法:通过定点突变技术将1.2拷贝HBV野生型质粒p3.8Ⅱ构建成核壳蛋白变异株V60和L97.经序列测定和生物学活性检测后,野生株和变异株重组质粒分别亚克隆入EB病毒表达载体EBO-plpp以稳定表达.重组载体EBO-野生株、EBO-V60和EBO-L97分别作内切酶双酶切和序列测定鉴定,再用脂质体介导转染HepG2细胞,ELISA(Abbott)试剂盒定量检测各株培养上清HBV抗原,转染细胞用FITC标记的鼠抗HLA-ABC单抗染色,流式细胞术分析细胞表面HLA-I表达.结果:3株重组载体经内切酶消化,电泳后显示2条区带,分别与1.2拷贝HBV基因组和EBO载体大小相同.测序结果证实EBO-V60和EBO-L97分别在nt2078 C→G和nt2189 A→C,保持原定点突变.EBO-野生株的培养上清HBeAg定量S/CO值明显高于变异株V60和L97,3株HBsAg定量S/N值接近,HBsAg表达相近表明实验的转染效率相当.EBO空载体转染的HepG2细胞HLA-I轻微表达,3株重组载体转染细胞HLA-I的荧光强度不同,野生株增强为18.2,L97明显升高至34.5,而V60降低至3.4.结论:HBV能增强HepG2细胞表面HLA-I/抗原肽复合物的表达,核壳蛋白热点变异V60和L97可使宿主细胞HLA-Ⅰ表达发生变化.     

乙型肝炎病毒核心蛋白突变体干扰HBV包装的实验研究

目的：构建乙型肝炎病毒（HBV）核心蛋白突变体基因的真核表达载体，转染HepG2细胞，观察其表达及干扰HBV颗粒包装的显性负调节作用。方法：采用PCR从质粒pHBVadrl-A1中扩增HBVadr1-A1中扩增HBV C基因和S基因，分别克隆到pGEM-T载体上，构建成pGEM-T-C和pGEM-T-S，进而构建成pGEM-T-CS载体，用HindⅢ切出克隆基因片段与pcDNA3.1连接，经PCR鉴定后构建成真核表达载体pcDNA3.1^ -CS,DNA测序显示基因融合正确，表达载体转染HepG2细胞，经G418筛选得到高拷贝转化子，逆转录－聚合酶链反应（RT－PCR）检测重组蛋白体外表达，用HBV阳性血清感染HepG2细胞，72h后提提细胞内HBV DNA，斑点杂交法分析各组DNA，结果：核心蛋白突变体在HepG2细胞内得到表达，且表达了该重组蛋白的HepG2细胞内HBV DNA量不同程度地低于对照组，表明重组蛋白具有抗HBV包装的DN突变体作用，结论：HBV核心蛋白与表面蛋白融合基因的真核表达载体pcDNA3.1-CS能够体外表达核心蛋白突变体，该突变体具有干扰HBV颗粒包装的显性负调节作用。     

乙型肝炎病毒基因组剪接变异体结构分析

目的 了解乙型肝炎病毒(HtBV)基因组剪接变异体的基因结构及特点。方法 从慢性乙型肝炎患者血清中扩增并克隆HBV基因组剪接变异体DNA，测序并比较基因结构特点。结果 共获得10种HBH基因组剪接变异体，基因组大小介于765～2039bp之间。导致剪接变异体产生的5′端供体位点和3′端受体位点各6个。HBVH基因组剪接变异体在C、前-Sl、前-S2及S编码区存在不同程度的缺失，但均保留与致病密切相关的X基因以及病毒复制、包装所必须的DNA序列。结论 HBV基因组剪接变异体在慢性乙型肝炎患者血清中普遍存在。     

Development of fulminant hepatitis B (precore variant mutant type) after the discontinuation of low‐dose methotrexate therapy in a rheumatoid arthritis patient

A 75‐year‐old female rheumatoid arthritis patient who was positive for hepatitis B surface antigen and for antibodies to hepatitis Be antigen showed liver dysfunction, and therefore methotrexate (MTX) therapy was discontinued. Her drug lymphocyte stimulation test indicated positivity for MTX. Her liver dysfunction improved briefly, but she developed fulminant hepatitis with elevated levels of hepatitis B virus (HBV)/DNA polymerase and subsequently died. HBV/DNA analysis performed with polymerase chain reaction–mutation site–specific assay revealed that the fulminant hepatitis was caused by a precore mutant virus. Sudden reactivation of the immune system by discontinuation of MTX may have led to the attack on infected cells. Even when hepatitis Be antibodies are present, MTX should not be used in patients who have chronic infection with HBV.     

Fulminant hepatitis related to transmission of hepatitis B variants with precore mutations between spouses.

A precore defective variant of hepatitis B virus has been indicated to cause fulminant hepatitis in various instances such as intrahospital outbreaks or mother-to-child transmission of hepatitis B virus. To learn whether similar variants are involved in interspouse transmission, we analyzed three cases of fulminant hepatitis B that developed in formerly healthy subjects whose only exposure to hepatitis B virus was contact with their longtime spouses, who were carriers of HBV and positive for antibody to HBe. The DNA clones for precore and S genes were propagated from patients and spouses and sequenced. Because of the conservation of S-gene sequences and the identity of subtypes between patient and spouse, it was suggested that patients were infected with hepatitis B virus from their spouses, not from other sources. A TGG-to-TAG mutation at the 28th codon of the precore gene of hepatitis B virus was commonly observed in all DNA clones from patients with fulminant hepatitis and from their spouses. A 29th-codon GGC-to-GAC mutation was additionally evident in DNAs from one patient-and-spouse couple. A significant rise in the circulating hepatitis B virus concentration was transiently observed in the index spouse of this case just before development of fulminant hepatitis in her husband. The increase in circulating HBV DNA was associated with a rise in abundancy of variants with mutations at both the 28th and 29th codons, compared with variants with only a 28th-codon mutation. The double mutation in hepatitis B virus DNA may either help the virus escape immune surveillance or replicate at a higher rate than before.     

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5 terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.     

Development of fulminant hepatitis B (precore variant mutant type) after the discontinuation of low-dose methotrexate therapy in a rheumatoid arthritis patient

A 75-year-old female rheumatoid arthritis patient who was positive for hepatitis B surface antigen and for antibodies to hepatitis Be antigen showed liver dysfunction, and therefore methotrexate (MTX) therapy was discontinued. Her drug lymphocyte stimulation test indicated positivity for MTX. Her liver dysfunction improved briefly, but she developed fulminant hepatitis with elevated levels of hepatitis B virus (HBV)/DNA polymerase and subsequently died. HBV/DNA analysis performed with polymerase chain reaction-mutation site-specific assay revealed that the fulminant hepatitis was caused by a precore mutant virus. Sudden reactivation of the immune system by discontinuation of MTX may have led to the attack on infected cells. Even when hepatitis Be antibodies are present, MTX should not be used in patients who have chronic infection with HBV.     

Genomic heterogeneity of hepatitis B virus (HBV) and outcome of perinatal HBV infection

BACKGROUND/AIMS: Data regarding hepatitis B virus (HBV) genomic heterogeneity in perinatal infection are incomplete, although HBV variants might be involved in neonatal fulminant hepatitis (ALF). We investigated HBV variability in infected babies showing different clinical courses. METHODS: We analyzed HBV genomes isolated from nine vertically infected babies and the mothers of four of them. Two infants born to HBe-antigen (HBeAg)-positive women developed a chronic infection; seven babies (six born to anti-HBe mothers) developed acute hepatitis that had a fulminant course in four cases and a benign course in three. Two babies developing ALF received anti-HBV immunoprophylaxis at birth. RESULTS: Viruses carrying no significant mutation infected infants born to HBeAg-positive women. HBeAg-defective viruses were detected both in children with benign and fulminant hepatitis and their mothers. A double nucleotide mutation at positions 1762 and 1764 of the HBV core-promoter was found in two of the four infants with ALF, although it was not detected in isolates from the mother of one of them. No significant S gene mutation was found in HBV from any of the babies. CONCLUSIONS: This study indicates that HBV genomic heterogeneity is not primarily involved either in the evolution of the infection or the failure of neonatal HBV immunoprophylaxis.     

Fulminant hepatitis after allogenic bone marrow transplantation caused by reactivation of hepatitis B virus with gene mutations in the core promotor region

Under immunosuppressive conditions after hematopoietic stem cell transplantation (HSCT), even if hepatitis B virus (HBV) antigen is negative but hepatitis B surface antibody (HBsAb) or hepatitis B core antibody (HBcAb) is presented, HBV reactivates and sometimes causes fulminant hepatitis. However, it remains unclear which patients will develop fulminant hepatitis, or whether fulminant hepatitis is caused by host-related factors or by virus-related factors. A 30-yr-old man with a history of aplastic anemia since 3 yr of age underwent allogenic BMT, when HBsAb and HBcAb were positive but HBs antigen (HBsAg) was negative. The donor was negative for HBsAg, HBsAb and HBcAb. After transplantation, the patient was complicated by acute graft-vs.-host disease (GVHD), cytomegalovirus infection, intestinal thrombotic microangiopathy and aspergillus colitis. Chronic GVHD was well controlled by FK506 and prednisolone. Twenty months after transplantation, the patient was admitted with general fatigue and liver dysfunction and was found to be positive for HBsAg and HBeAg. His serum HBV-DNA level was >8.8 log of the genome equivalent (LGE)/mL. Therefore, he was diagnosed as having hepatitis B caused by HBV reactivation and 100 mg/d lamivudine treatment was started. However, jaundice and hepatic failure deteriorated and became fatal. On analysis of the HBV-DNA, two adjacent gene mutations in the core promoter region (T1762/A1764) were detected. Increased replication of the mutated HBV might have caused HBV reactivation which progressed to fulminant hepatitis.     

Association of hepatitis B virus subgenotypes and basal core promoter/precore region variants with the clinical features of patients with acute hepatitis

BACKGROUND: In endemic areas, including Japan, basal core promoter (BCP) and precore (PC) variants of hepatitis B virus (HBV) have been reported to be associated with the clinical outcome of acute hepatitis B patients. However, the associations of BCP/PC variants with clinical outcomes have not been observed in nonendemic areas. HBV subgenotypes, which show geographic variations in prevalence, may underlie this discrepancy in clinical outcomes. Little is known about the differences in the clinical and virological features of HBV subgenotypes and BCP/PC variants. The aim of this study was to investigate the distributions of subgenotypes and BCP/PC variants to identify clinical differences in acute hepatitis B patients. METHODS: One hundred thirty-nine patients with acute hepatitis were enrolled. Nested polymerase chain reaction was used to amplify the pre-S region of HBV for genotyping and the BCP/PC regions for variant screening. RESULTS: HBV subgenotypes A1 (n = 3), A2 (n = 28), B1 (n = 3), B2 (n = 9), C1 (n = 5), C2 (n = 84), C variant (n = 1), D2 (n = 3), and H (n = 3) were detected. BCP/PC variants were not associated with progression to chronic hepatitis. Patients infected with subgenotype C2 who progressed to fulminant hepatic failure frequently carried variants at nucleotides non-T1753 and non-T1754 and T1762, A1764, and A1896. CONCLUSIONS: BCP/PC variants would be associated with progression to fulminant hepatitis in subgenotype C2. Knowledge of HBV subgenotypes and BCP/PC variants is useful for developing strategies to treat acute hepatitis B patients.     

慢性HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应（PCR）－限制性片段长度多态性（RFLP）分析与荧光定量聚合酶链反应检测HBVDNA相结合,对30例HBsAg（＋）、抗－HBe( )及抗－HBc( )慢性HBV感染者,其中无症状携带者（AsC)9例、慢性乙型肝炎（CHB）12例及慢性重症肝炎（CHF）9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组3例（33．33％）,CHB组9例（75％）及CHF组8例（88．89％）有A83(nt1896)变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异与HBV不同感染状态中都可见,其病毒复制水平与肝病活动相关。     

Fatal Fulminant Hepatitis B after Withdrawal of Prophylactic Lamivudine in Hematopoietic Stem Cell Transplantation Patients

Hepatitis B virus (HBV) reactivation can give rise to acute hepatitis and even fatal fulminant hepatitis in patients receiving immunosuppressive or cytostatic treatment. Recently, the prophylactic use of lamivudine for HBV reactivation in HBV surface antigen-positive chronic-disease patients undergoing hematopoietic stem cell transplantation (HSCT) has been reported. However, the appropriate duration for this prophylactic therapy is unclear. Here, we report 2 cases of fatal fulminant hepatitis B reactivation in HSCT patients after lamivudine withdrawal. One patient with non-Hodgkin's lymphoma completed 6 courses of CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine [Oncovin], and prednisone) and autologous peripheral blood SCT (PBSCT). Lamivudine was discontinued 3 months after transplantation. The second patient had acute myeloid leukemia. He received induction chemotherapy and postremission allogeneic PBSCT as late intensified consolidation therapy. Lamivudine treatment was discontinued 10 months after transplantation. In both patients, HBV reactivation 2 to 3 months following lamivudine cessation led to fatal fulminant hepatitis. We suggest that the duration of prophylactic use of lamivudine in chronic HBV carriers receiving HSCT be prolonged until the patient's immune system has been reconstituted.