乙型肝炎核心抗原为载体进行丙型肝炎混合性治疗疫苗的实验

目的 以HBcAg为载体进行丙型肝炎混合性治疗疫苗的实验研究。方法 利用DNA重组技术将HCVT表位（131～140位氨基酸）和（1445～1453位氨基酸）分别插入HBcAg el—loop处，构建病毒颗粒样抗原表达载体pTrc-core-T1和pTrc-core-T2，并在大肠埃希菌DH5α中表达，蔗糖密度梯度离心提取纯化表达产物HBcAg—T1和HBcAg—T2。以T1和T2表位肽混合物、HBcAg—T1和HBcAg-T2混合蛋白免疫Balb／C小鼠，并设空白对照，适时行抑瘤实验；流式细胞仪检测CD4^＋、CD8^＋、IL-4及IFN-γ、IL-5，ELISA法检测IL-12；并行细胞杀伤实验，以观察混合蛋白的免疫原性。结果 PCR法鉴定质粒pTrc—core—T1和pTrc—core—T2正确。抑瘤实验中HBcAg—T1和HBcAg—T2混合蛋白免疫组仅1只小鼠形成瘤块，直径0．1cm，低于对照组的平均直径1．3cm、T1T2混合肽免疫组的0．9cm；混合蛋白免疫组脾细胞内CD8^＋T细胞占（20．21±2．01）％，高于混合肽免疫组的（15．33±1．45）％和空白对照组的（5．09±1．66）％（P〈0．01）；3组内脾细胞内IFN-γ阳性细胞百分比分别为（1．58±0．05）％、（0．88±0．02）％和（0．53±0．03）％（P〈0．01）；血清中IL-5较混合表位肽组有所下降（P〈0．01）；而ELISA检测混合蛋白组IL-12高于T1T2混合表位肽免疫组；混合蛋白免疫组小鼠HCV特异细胞毒性T淋巴细胞（CTL）活性明显高于T1T2混合表位肽免疫组（P〈0．01）。结论 HBcA乎T1和HBcAg～T2混合蛋白诱导出高水平的细胞免疫应答，可作为HCV治疗性疫苗的候选。     

应用共同抗原的多克隆与单克隆抗体进行肠道病毒诊断的研究

目的制备肠道病毒可能的共同抗原多克隆与单克隆抗体,用于早期诊断肠道病毒。方法RT-PCR扩增VP1N端122氨基酸这一可能的共同抗原基因片段,连接至PET-30a表达载体,转化到大肠杆菌BL21(DE3)中进行体外表达,表达重组蛋白经Western-blot活性鉴定后用电洗脱法进行纯化。纯化蛋白免疫昆明鼠获取多克隆抗体,多克隆抗体用饱和硫铵沉淀法进行纯化。纯化蛋白免疫BALB/c鼠,用杂交瘤技术进行单克隆抗体制备,并用Protein A/G亲和柱进行纯化。制备的抗体用间接免疫荧光法(IFA)检测肠道病毒。结果重组蛋白经Western blot检测,能被国外商品化的肠道病毒组特异性的5-D8/1单克隆抗体、含脊髓灰质炎病毒抗体的人血清与分别代表肠道病毒A、B、C三组的单型血清所识别。制备其相应的多克隆和单克隆抗体,与5-D8/1单克隆抗体同时应用间接免疫荧光法(IFA)检测29株(27型)福建分离株病毒抗原,显示:制备的多抗或单抗的检出率比5-D8/1单抗高约10%~40%。结论结合福建省当地株制备肠道病毒共同抗原的抗体检测变异较大的当地株,更具优势。     

鼠疫耶尔森菌LcrV抗原B细胞表位的预测

目的预测鼠疫耶尔森菌LcrV抗原的B细胞表位,为抗LcrV单克隆抗体的制备提供理论依据,探索适合我国鼠疫耶尔森菌蛋白B细胞表位组研究的可行方案。方法采用EMBOSS网络平台以及DNAStar Protean软件分析LcrV蛋白的二级结构、可塑性、亲水性和抗原性指数等参数,进行B细胞表位的预测和分析。结果LcrV蛋白的B细胞表位可能位于第16～24、26～31、45～50、175～181、208～213和280～289位等氨基酸区域内或附近,其中16～24、26～31、175～181、208～213等4个短肽都与文献报道相吻合。结论预测得到的表位为后续抗LcrV抗原单克隆抗体的制备奠定了理论基础,为进一步表位分析和鉴定提供了参考依据;预测结果的准确性高,有助于利用表位预测法建立鼠疫菌蛋白B细胞表位研究的平台。     

嗜人按蚊中肠单克隆抗体的制备及初步鉴定

目的 制备并鉴定抗嗜人按蚊中肠单克隆抗体杂交瘤细胞株。方法 用嗜人按蚊中肠做抗原，用细胞融合技术制备单克隆抗体，并采用ELISA法对单克隆抗体进行筛选和鉴定。结果 建立了1株持续分泌抗嗜人按蚊中肠单克隆抗体的杂交瘤细胞株。BALB／c小鼠腹水单克隆抗体的最高稀释度达1：12800；单克隆抗体的重链属IgG1亚类；Western blot证实单抗能与嗜人按蚊中肠抗原特异性结合。结论 成功制备了抗嗜人按蚊中肠单克隆抗体。     

抗肺炎衣原体蛋白酶样活性因子单克隆抗体在冠状动脉硬化患者肺炎衣原体感染诊断中的初步应用

目的 制备抗肺炎衣原体蛋白酶样活性因子重组蛋白的单克隆抗体,检测冠状动脉硬化患者的外周血单核细胞和冠状动脉瘤斑块中肺炎衣原体特异性抗原.方法 以肺炎衣原体蛋白酶样活性因子重组蛋白免疫小鼠,与SP2/0细胞融合后,采用间接ELISA和Western blot筛选阳性杂交瘤细胞,经亚克隆建株;小鼠体内诱生腹水法制备单克隆抗体,用硫酸铵沉淀法对腹水中的单克隆抗体进行纯化,测定其亚类和效价;检测肺炎衣原体标准株以鉴定其特异性.建立间接ELISA方法检测冠状动脉硬化患者外周血单核细胞和冠状动脉瘤斑块标本中的肺炎衣原体抗原.结果 获得4株稳定分泌抗蛋白酶样活性因子重组蛋白的杂交瘤细胞株,分别命名为3F8、7B9、8C4和11B5,其效价分别为1∶28000、1∶16000、1∶38000和1∶12000;经亚类鉴定,3F8和7B9杂交瘤细胞分泌的单克隆抗体为IgG2b,其它2株均为IgG1.以制备的单克隆抗体建立间接ELISA检测肺炎衣原体抗原,与经典PCR方法的检测结果比较,检出率具有较高的一致性.结论 成功获得了抗肺炎衣原体蛋白酶样活性因子重组蛋白的特异性单克隆抗体,能特异地识别天然肺炎衣原体蛋白酶样活性因子抗原,有望应用于冠状动脉硬化患者肺炎衣原体感染的抗原诊断.     

抗广州管圆线虫Ⅴ期幼虫55ku抗原单克隆抗体的制备、鉴定及初步应用

目的制备抗广州管圆线虫Ⅴ期幼虫55 ku抗原单克隆抗体,初步应用于大鼠广州管圆线虫感染检测。方法利用Western blot鉴定能被感染广州管圆线虫Ⅴ期幼虫大鼠血清识别的特异性抗原,经电渗法获得抗原后免疫BALB/c小鼠;取脾细胞,采用常规细胞融合方法建立筛选分泌高滴度、高特异性单克隆抗体杂交瘤细胞株,用ELISA、Western blot对获得的杂交瘤细胞株进行鉴定,用生产的单克隆抗体以抗体夹心ELISA法检测感染广州管圆线虫Ⅴ期幼虫大鼠血清循环抗原水平。结果经Western blot鉴定,得到能被感染广州管圆线虫Ⅴ期幼虫大鼠血清识别的广州管圆线虫Ⅴ期幼虫55 ku抗原;获得2株分泌高滴度抗广州管圆线虫Ⅴ期幼虫55 ku抗原的单克隆抗体杂交瘤细胞株,命名为ACV1和ACV2,其分泌的抗体均为IgG2b亚型,均能识别广州管圆线虫Ⅴ期幼虫55 ku抗原,与日本血吸虫抗原等其他寄生虫抗原无交叉反应。用该单抗以ELISA方法检测感染广州管圆线虫Ⅴ期幼虫大鼠血清循环抗原,阳性率为73.3%(22/30)。结论制备的抗广州管圆线虫Ⅴ期幼虫55 ku抗原杂交瘤细胞株能分泌高滴度、高特异性的单克隆抗体,该单抗可应用于广州管圆线虫Ⅴ期幼虫循环抗原的检测。     

日本血吸虫病诊断分子B细胞表位的预测与鉴定

目的应用生物信息学技术对日本血吸虫病常用诊断分子进行B细胞表位的预测,筛选出具有潜在诊断价值的表位分子,采用基因工程方法制备目的表位的融合蛋白并进行抗原性鉴定.方法将日本血吸虫膜蛋白22 kDa(Sj22)、膜蛋白23 kDa(Sj23)、信号蛋白14-3-3(Sj14-3-3)、谷胱甘肽S转移酶(Sj26)分子的全基因序列输入BioSun软件的工作区,经多参数比较筛选可能的B细胞表位,克隆、表达预测表位,用表达的融合蛋白作为抗原与血吸虫病人血清反应,以筛选和鉴定具有抗原性的表位.结果经预测分析,Sj22的B细胞表位可能在56～62位和127～133位氨基酸区域,Sj23的B细胞表位可能在149～156位和160～167位氨基酸区域,Sj14-3-3的B细胞表位可能在118～225位和130～137位氨基酸区域,Sj26的B细胞表位可能在143～149位和191～197位氨基酸区域.克隆、表达并纯化这8个表位的融合蛋白,经SDS-PAGE和Western blot检测抗原性,筛选出分别来自于Sj22、Sj14-3-3、Sj26的3条具有较强抗原性的表位片段.结论生物信息学技术结合分子生物学方法可用于筛选具有潜在诊断价值的表位.     

抗弓形虫水通道蛋白多肽抗体的制备及鉴定

目的 研制抗弓形虫水通道蛋白(TgAQP)的特异性多肽抗体,并应用于检测弓形虫ME49株中水通道蛋白的表达和亚细胞定位。方法 根据TgAQP氨基酸序列设计B细胞抗原多肽,并将合成的多肽与KLH偶联作为免疫原免疫新西兰大耳白兔制备多克隆抗体,通过ELISA、Western blot和免疫荧光的方法对所得抗体进行鉴定。结果 选定并合成抗原表位多肽,与KLH偶联作为免疫原制备多克隆抗体;经ELISA测定其多肽抗体效价达1∶40 000; Western blot表明制备的抗体能特异地识别天然弓形虫中29.9 kDa处的条带,与预测的TgAQP相对分子量大小相符;免疫荧光检测到抗血清识别的蛋白定位于弓形虫胞质中。结论 制备了抗弓形虫水通道蛋白多肽的多克隆抗体,为进一步研究弓形虫水通道蛋白的生物学功能和代谢特点奠定了基础。     

我国主要乙型肝炎病毒流行株中C抗原18～27位细胞毒性T淋巴细胞表位特点分析

目的 探讨我国HBV感染者中人类白细胞抗原(HLA)-A*0201限制性特异性细胞毒性T淋巴细胞(CTL)表位HBcAg(18～27)V/I变异体的病毒学背景特点.方法 应用生物信息学方法分析GenBank中30条不同HBV基因型的病毒株HBcAg(18～27)区段的序列特点.设计错配引物,构建针对HBV B、C基因型HBcAg(18～27)V/I的PCR限制性片段长度多态性分析(RFLP)筛选方法.以PCR-RFLP和测序方法检测来自我国8省区160份HBV基因型B、C标本的HBcAg(18～27)V/I基因特点.用表位预测网站分析不同HBcAg(18～27)特异性CTL表位基因变异体与HLA-A*0201分子结合力的差异.结果 成功构建了针对HBV基因B、C型HBcAg(18～27)V/I的PCR RFLP筛选方法;在调查的HBV B、C基因型血清中发现HBcAg(18～27)V的阳性率仅为1.88%(3/160);生物信息学分析显示HBcAg(18～27)I与HLA-A*0201分子的结合力明显低于HBcAg(18～27)V(HLA Ligand评分123与156,SYFPEITHI评分22与24).结论 我国流行的主要HBV流行株中HBcAg(18～27)的最后一位氨基酸主要表现为异亮氨酸,而非缬氨酸;在进行细胞免疫学研究时,以HBcAg(18～27)V作为参照需要充分考虑这一病毒学背景.     

禽流感病毒M1蛋白型特异性表位分析

目的对流感病毒基质蛋白1(M1)型特异性表位进行定位研究。方法采用针对禽流感病毒M1蛋白的型特异性单克隆抗体,淘选M13噬菌体展示的12肽随机肽库,进行M1蛋白表(拟)位分析。采用ELISA、竞争性ELISA、免疫印迹分析不同噬菌体拟位与单克隆抗体的免疫反应性。结果筛选获得共有序列NxPHLR,定位于M1蛋白222-224位(HPN)、229-230位(LR)氨基酸区域。含有NxPHLR基序的噬菌体拟位能与单克隆抗体发生特异性结合,且此结合能被天然病毒抗原抑制或阻断,表明拟位多肽真实模拟了天然病毒蛋白与单克隆抗体结合的抗原决定簇或表位。结论M1蛋白222-230位氨基酸(HPNSSARLR)序列构成了流感病毒型特异性表位。     

Treatment of chronic woodchuck hepatitis virus infection in the Eastern woodchuck (Marmota monax) with nucleoside analogues is predictive of therapy for chronic hepatitis B virus infection in humans

The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a model of hepatitis B virus (HBV)-induced disease. Several published studies have used this experimental animal model system to demonstrate potential antiviral therapies for chronic HBV infections. However, there has been little comparative information available on compounds used in clinical anti-HBV studies in WHV-infected woodchucks, thereby making interpretations of the potential relative effectiveness of new antiviral agents in humans more difficult. In this report, using a series of placebo-controlled studies, we compared the relative effectiveness of several nucleoside analogues that have been used in clinical trials for the treatment of chronic HBV infection against WHV replication in chronically infected woodchucks. Adenine-5'-arabinoside monophosphate (Ara-AMP [vidarabine]), ribavirin, (-)beta-L-2',3'-dideoxy-3'-thiacytidine (3TC [lamivudine]), and famciclovir (oral prodrug of penciclovir) induced depressions in viremia and intrahepatic WHV-DNA replication that were consistent with their relative effectiveness in anti-HBV human clinical trials. As observed in HBV-infected patients, 3' azido-3'-deoxythymidine (AZT [zidovudine]) had no effect on WHV replication in these studies. These experimental results more firmly establish chronic WHV infection in woodchucks as an accurate and predictive model for antiviral therapies against chronic HBV infection in humans and provide a baseline for comparative antiviral effects of other experimental antiviral agents in the WHV/woodchuck model system.     

Antiviral activity of clevudine [L-FMAU, (1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl) uracil)] against woodchuck hepatitis virus replication and gene expression in chronically infected woodchucks (Marmota monax)

L: -FMAU [1-(2-fluoro-5-methyl-beta,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of hepatitis B virus (HBV) and duck hepatitis B virus replication in cell culture and duck hepatitis B virus replication in acutely infected Peking ducks. The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of woodchuck hepatitis virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection.     

Induction of antibodies to the PreS region of surface antigens of woodchuck hepatitis virus (WHV) in chronic carrier woodchucks by immunizations with WHV surface antigens

BACKGROUND/AIMS: One goal of therapeutic vaccinations against chronic hepatitis B virus infection is to stimulate the B-cell responses to viral surface antigens in chronic carriers. Here we investigated the induction of antibody responses to hepadnaviral surface antigens in the woodchuck model, with emphasis on the vaccination of woodchucks chronically infected with woodchuck hepatitis virus (WHV). METHODS: Naive and chronically WHV-infected woodchucks were immunized with plasma-derived WHV surface antigens (p-WHsAg) containing the S and PreS sequences. Antibody responses to WHsAg and the WHV PreS region and viral load in immunized woodchucks were monitored. RESULTS: After repeated immunizations with WHsAg, 17 of 18 chronic WHV carriers developed a persistent antibody response to WHsAg. These antibodies were mainly directed to epitopes within the PreS region and detectable by Western blotting. However, neither WHV DNA nor WHsAg concentrations in these woodchucks changed significantly by immunizations and during the follow up. Sequence analysis of WHV genomes showed that no WHV mutants emerged after the induction of anti-WHs/anti-WHpreS antibodies. No immunopathological changes in livers of immunized animals were recognized thus far. CONCLUSIONS: Our study demonstrated that the immunological unresponsiveness of chronically WHV-infected woodchucks to WHsAg can be partially overcome by repeated immunizations with WHsAg.     

Gamma-interferon production in response to hepatitis B core protein and its synthetic peptides in patients with chronic hepatitis B virus infection.

The nucleocapsid of hepatitis B virus (HBV) is an efficient immunogen in activating T cells to produce interferon-gamma (IFN-gamma) in patients with chronic HBV infection. We investigated hepatitis B core antigen (HBcAg)-specific T cell recognition, which seems to be implicated in the pathogenesis of chronic liver disease. IFN-gamma production by peripheral-blood mononuclear cells of patients with chronic HBV infection [25 patients with chronic active hepatitis (CAH) and 14 asymptomatic carriers of HBV (ASCs)] was measured by an enzyme-linked immunosorbent assay. P19 polypeptide, which is derived from recombinant HBcAg particle (rHBcAg), increased IFN-gamma production in patients with CAH, but its effect was weaker than that of rHBcAg. P19 had no stimulating effect on T cells from ASCs. The fine specificity of T cell recognition of HBcAg was examined using 8 kinds of synthetic peptides. T cells from the patients who responded against P19 polypeptide recognized the sites within the common sequences of HBcAg and HBeAg (p72-90, P90-99, P108-122 and P126-146). These results suggest that HBcAg and P19 are cross-reactive at the T cell level, and that these T cells recognize the sites within the common sequences of HBcAg and HBeAg in HBV-infected patients.     

MxA inhibits hepatitis B virus replication by interaction with hepatitis B core antigen

Human MxA, an interferon-inducible cytoplasmic dynamin-like GTPase, possesses antiviral activity against multiple RNA viruses. Recently, MxA has also been demonstrated to have activity against the hepatitis B virus (HBV), a well-known DNA virus responsible for acute and chronic liver disease in humans. We investigated the molecular mechanism for the anti-HBV activity of MxA. Our results demonstrated that in HepG2.2.15 cells, MxA GTPase independently suppressed the production of hepatitis B surface antigen and HBV DNA without changing the level of hepatitis B core antigen (HBcAg) and the distribution of HBV mRNA. MxA significantly reduced the level of the encapsidated pregenomic RNA. Through its central interactive domain, MxA interacted with HBcAg, causing accumulation of the proteins in perinuclear compartments. MxA-HBcAg interaction significantly affected the dynamics of HBcAg by immobilizing HBcAg in the perinuclear structures. CONCLUSION: MxA displays antiviral activity against HBV involving a mechanism of MxA-HBcAg interaction that may interfere with core particle formation.     

Immunosuppression reactivates viral replication long after resolution of woodchuck hepatitis virus infection

Resolution of hepatitis B virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with woodchuck hepatitis virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such woodchucks resulted in transient reactivation of WHV replication. Serum of 1 woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual virus. CONCLUSION: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection.     

乙型肝炎病毒核心蛋白在HepG2.2.15细胞中的亚细胞定位及转移

目的观察HepG2．2．15细胞内HBV核心蛋白的亚细胞定位及转移，了解核心蛋白的入核机制。方法2％二甲亚砜或（和）1μmol／LBay414109处理HepG2．2．15细胞4d；荧光共聚焦显微镜观察HBsAg和HBcAg在细胞内的定位；Westernblot检测胞质和胞核中的HBcAg水平；选择性PCR检测胞核内HBV共价闭合环状DNA（cccDNA）水平。结果二甲亚砜处理提高了胞质和胞核内的HBcAg表达及核内的cccDNA水平；Bay414109处理后胞质中HBcAg水平下降但胞核中HBcAg水平上升，cccDNA水平下降；联合应用二甲亚砜和Bay41—4109处理后HBsAg在胞质内呈条索状分布，胞质中HBcAg水平下降，但胞核内HBcAg明显上升，cccDNA水平下降。结论HepG2．2．15细胞中存在HBV核心颗粒入核障碍，游离核心蛋白易于通过核孔，二甲亚砜可促进核心蛋白进入细胞核，并有助于cccDNA的形成。     

Liver-targeted antiviral nucleosides: Enhanced antiviral activity of phosphatidyl-dideoxyguanosine versus dideoxyguanosine in woodchuck hepatitis virus infection in vivo

It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3'-Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl- dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP- ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP- ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis. (Hepatology 1996 May;23(5):958-63)     

Isolation, characterization, and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus.

The human hepatitis B virus (HBV) and the woodchuck hepatitis virus (WHV) are closely related by several criteria and belong to the same class of DNA viruses. The DNA genomes from these viruses are difficult to obtain in quantities required for biochemical analysis. We have, therefore, cloned these two DNAs in the vector lambda gtWES and subcloned into the kanamycin resistance plasmid pA01. Comparison of the recombinant DNAs with authentic viral DNAs by specific hybridization, size, and restriction enzyme analysis suggests that the recombinants contain the complete genome of each virus. The nominal size of the cloned HBV genome was 3150 base pairs, compared to 3200 base pairs for the cloned WHV genome. The small amount of nucleic acid homology previously reported between the HBV and WHV DNAs could be demonstrated between the cloned viral DNAs.