Hepatitis B polypeptide vaccine preparation in micelle form

The immunoprophylaxis of hepatitis B is hampered by the lack of a technique for growing hepatitis B virus (HBV) in tissue culture. Plasma from persistently infected individuals, one source of viral antigen, contains characteristic 22-nm spherical particles which share a common antigen (the hepatitis B surface antigen or HBsAg) with the outer envelope of the 42-nm double-shelled DNA virus. Highly purified inactivated 22-nm particles have been shown to be safe and to confer protective immunity against HBV in a recent large-scale clinical trial. We have already described the extraction from the particles of a complex of two proteins which are antigenic determinants of HBV--the polypeptide with molecular weight (MW) between 22,000 and 24,000 (called p23) and the glycosylated polypeptide (called gp28) with MW in the range 26,000--29,000 which is thought to be the glycosylated form of p23. We now report the preparation from this complex of water-soluble protein micelles which may be a suitable basis for a second-generation hepatitis B vaccine.     

Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma

Hepatitis B virus (HBV) may be one of the agents involved in the aetiology of human primary liver cancer. This hypothesis is supported by (1) the similarity between the geographical distribution of chronic carriers of the viral surface antigen (HBsAg) and that of hepatocellular carcinoma (HCC); (2) the increase in the prevalence of HBV markers in serum of patients with primary liver cancer when compared with the general population; (3) the observation that HBV infection precedes the development of the tumour. Moreover, these epidemiological indications of an association between HBV infecton and hepatocellular carcinoma are supported by the detection of HBV markers such as HBsAg or viral DNA sequences, although in a non-integrated form in tumour tissue. To study the relationship between HBV and primary liver cancer further, we looked for the presence of free or integrated viral DNA in tumour tissue of human hepatocellular carcinomas and in a HBsAg-producing human hepatoma cell line. Using the blot-transfer hybridization technique and cloned HBV DNA as a probe, we have now demonstrated that the viral DNA is integrated in the cellular genome both in tumour tissue and in a hepatoma cell line.     

治疗性乙型肝炎疫苗研究反思

 “治疗性乙肝疫苗”是以乙肝病毒(HBV)的表面抗原(s抗原)为基础的生物制剂,目的是激发抗s抗原免疫反应,终止HBV慢性感染.HBV的e抗原与s抗原无抗原性关联,对e抗原的反应也与病毒及感染细胞的清除无关,因此以II期临床试验者血清有关病毒e抗原的数据结果,尚不能判断“治疗性乙肝疫苗”是否有效.疫苗的应用是抵御病毒入侵,而治疗性乙肝疫苗是在病毒已经进入人体后应用,在患者肝细胞可能广泛受累的情况下,疫苗一旦打破耐受激发抗s抗原免疫反应,除能清除血清中游离的病毒和s抗原颗粒外,将直接攻击被感染的肝细胞.由于无法估计慢性HBV感染者肝细胞感染程度,所以无法推测免疫反应发生后,免疫病理所致的肝损程度以及相应的风险.在应用上隐含如此风险,是“治疗性乙肝疫苗”走不出实验室的重要因素之一.     

RNA interference-mediated inhibition of Hepatitis B Virus replication

Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.     

Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line.

A significant aspect of primary hepatic carcinoma in man is the high positive correlation of hepatocellular carcinoma with infection with hepatitis B virus (HBV)1. Analysis of the relationship between HBV infection and oncogenesis is difficult because natural infection with HBV is limited to man and experimental infection has been achieved only in chimpanzees and gibbons. Furthermore, because HBV has not been successfully propagated in cell culture, basic study of virus-cell interaction of the aetiological agent of one of the most widespread infections of man has been impossible. Recently, however, a cell line (PLC/PRF/5) derived from a human hepatoma biopsy was described which produces the HRV surface antigen (HBsAg) and so provides a tool for the experimental investigation of HBV in viro. We now report the derivation and characterisation of two additional cell lines primary liver carcinomas. In contrast to the PLC/PRF/5 cell line, these cell lines retain the capacity to synthesise many human plasma proteins, including both albumin and alpha-fetoprotein (AFP). One of these lines also produces BHsAg. We also present evidence that HBsAg synthesis and secretion in this cell line are correlated with the growth state of the culture. This finding is in contrast to the continuous HBsAg production found in the PLC/PRF/5 cell line.     

Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen.

DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the surface antigens is a protein consisting of 226 amino acids and with a molecular weight of 25,398. The portion of the gene coding for this protein apparently contains no intervening sequences.     

依纽小单胞菌2-脱氧蟹肌醇合成酶基因的克隆与表达

从伊纽小单孢菌(Micromonospora inyoensis)扩增参与紫苏霉素生物合成的2-脱氧蟹肌醇合成酶基因sisB,并分别将其克隆到筛选载体pUC18和表达载体pET-30a上,其开放阅读框长1 185 bp,编码含394个氨基酸残基(41.94 ku)的多肽链,将pET-sisB转化大肠杆菌E.coliBL21(DE3),使sisB基因实现表达.基因sisB的碱基序列与棘孢小单孢菌(M.echinospora)的基因gntB的碱基序列的同源性高达93%.预测的SisB蛋白序列与GntB蛋白序列的同源性为95.2%.基因sisB是继紫苏霉素抗性基因srm1(AY661430)和参与紫苏霉素生物合成的糖基转移酶基因sisD(DQ250992)和sisZ(DQ250994)后,从依纽小单孢菌克隆到的又一新基因.该基因在GenBank的接受号为DQ250993.     

gag Gene of mammalian type-C RNA tumour viruses.

The translation product of the gag gene of mammalian type-c- RNA viruses is a 65,000-68,000 molecular weight precursor polypeptide (Pr65) whose cleavage leads to the formation of four virion proteins, p30, p15, p12 and p10. An immunological approach has been used to establish the arrangement of the sequences coding for these proteins within the viral genome as (5') p15-p12-p30-p10 (3').     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro- particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR- Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

乙型肝炎表面抗原基因在酵母中的表达——Ⅰ.HBsAg基因在酵母中的克隆

将乙型肝炎表面抗原基因组装进穿梭质粒,经E.coli扩增、鉴定后转入酵母细胞得到了转化子。经放射免疫分析,在此转化子中HBsAg未得到表达,可能是阅读框架不一致造成的。此外,我们对酵母DNA重组技术进行了摸索,并简化和改进了一些步骤。     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the microparticle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

在杂合启动子控制下乙肝病毒表面抗原SA-28融合基因在酵母中的表达及调控

采用酵母杂合启动子PADH2－CUP1或PADH2－GAPDH及终止子TADH1，构建了一系列酵母表达载体．在这些表达载体中插入乙肝病毒表面抗原S－preS1融合基因SA－28后，将合乙肝病毒表面抗原的表达单元克隆至高稳定质粒PHC11的BamHI位点．然后将表达质粒分别转化酿酒酵母Y16，Y19．对SA－28基因表达的研究表明，在酵母菌胞内实现了SA－28基因的高表达，且表达受葡萄糖浓度调控．     

HIV-1和HBV复合型DNA免疫的初步研究

近年的研究表明,在啮齿类和非人灵长类免疫带有编码病毒和细菌抗原基因的质粒ＤＮＡ可以激发体液和细胞免疫应答．在本实验中,将ＨＢＶ的Ｓ基因和ＨＩＶ－１的ｇｐ１６０基因以融合形式插入到载体ｐｃＤＮＡ３中,其能表达ＨＢｓＡｇ和ｇｐ１６０的融合蛋白,并将此质粒ＤＮＡ分别直接注射到Ｂａｌｂ／ｃ小鼠和Ｓｗｉｓ小鼠．三次免疫后,用ＥＬＩＳＡ的方法初步检测ＨＢｓＡｇ和ｇｐ１６０抗原特异的抗体免疫应答均为阳性．结果说明,带有ＨＢＶ和ＨＩＶ－１融合基因的质粒ＤＮＡ直接免疫小鼠后,均激发了小鼠产生相应的免疫应答反应,这个结果为研究和生产多价疫苗提供了新的思路     

Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype

Recent advances in molecular genetics have led to the possibility of using large DNA viruses, such as vaccinia virus, as a biological delivery system for immunizing man against unrelated disease-causing agents. When live vaccinia virus recombinants expressing the hepatitis B virus surface antigen (HBsAg), the influenza A virus haemagglutinin, the herpes simplex virus (HSV) type 1 D glycoprotein, the rabies virus G glycoprotein and the vesicular stomatitis virus G glycoprotein were used for immunization, animals were protected upon challenge with the appropriate pathogenic agent. A major concern with using such vaccines, however, stems from the previously documented vaccinia virus-associated post-immunizing complications. We present here experimental evidence that thymidine kinase-negative (TK-) vaccinia virus recombinants, constructed by inserting a variety of DNA coding sequences into the vaccinia virus tk gene, are less pathogenic for mice than wild-type virus.     

具全期启动子盒的杆状病毒高效表达载体的组建与应用

通过ＰＣＲ扩增，得到苜蓿丫纹夜蛾核型多角体病毒（ＡｕｔｏｇｒａｐｈａｃａｌｉｆｏｒｎｉｃａＮｕｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ，ＡｃＮＰＶ）具早晚期启动子元件的ｐ３５基因启动子，将其插入到杆状病毒转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３多克隆位点上游，使之与ｐＳＸＩＶＶＩ＋Ｘ３质粒中的人工合成后期启动子（ＰＳｙｎ）、多角体ＸＩＶ启动子（ＰＸＩＶ）串联构成早期、晚期、极晚期能持续启动外源基因表达的转移载体质粒ｐＳＸ３５．将ｐＳＸ３５用于组建含ＨＢｓＡｇ基因并形成多角体的重组ＴｎＮＰＶ，ＨＢ－ｓＡｇ基因的表达量显著提高，表达时间亦明显提前，从而实现了外源基因在杆状病毒表达系统的全期、高效表达．ｍＲＮＡ引物延伸试验结果显示，Ｐｐ３５在重组病毒中可产生２套转录本，分别于病毒感染的早期和晚期起始ＨＢｓＡｇ基因的表达．     

庆大霉素生物合成关键酶基因在E.coli中的克隆与表达

从庆大霉素产生菌?棘孢小单孢菌基因组中扩增出参与庆大霉素生物合成的关键酶基因——2-脱氧青蟹肌糖合成酶基因GntB,将其分别克隆到克隆载体pBS-T和表达载体pET-22b(+)上,并将pET-gntB转化大肠杆菌E.coliBL21(DE3),用IPTG诱导使GntB基因实现表达.GntB基因大小为1 193 bp,其编码的氨基酸含397个残基,约为42 kD的多肽链.