以牛痘病毒为载体的乙型肝炎病毒表面抗原基因的表达

乙型肝炎是一种严重危害人类健康的疾病,对它的防治国内外均十分重视。乙型肝炎是由乙型肝炎病毒(HBV)感染所致。HBV是部分单链的DNA病毒,其基因组编码了表面抗原蛋白、核心抗原蛋白以及内源DNA聚合酶等蛋白质。乙肝表面抗原蛋白(HBsAg)是病毒的外壳蛋白,在乙肝病人的血液中能形成直径约22nm的表面抗原颗粒。用它作为抗原给动物注射,可使动物产生专一性抗体。     

乙型肝炎病毒前S1抗原(1-42)与核心抗原(1-144)在大肠杆菌中表达的融合蛋白CS1的免疫原性研究

对重组乙型肝炎（乙肝）病毒前S1抗原（1－42）及核心抗原（1－144）表达的融合蛋白CS1进行了免疫原性的研究分析,以便为探索HBV治疗性疫苗地研制提供实验依据。用脂质体转染的方法转染小鼠肥大细胞瘤细胞系P815,用乳酸脱氢酶的方法检测了该融合蛋白诱导小鼠的细胞毒T淋巴细胞（CTL）反应,用ELISA方法测定了小鼠的体液免疫反应。结果表明,用脂质体转染的方法建立了表达乙肝核心和前S1抗原的细胞系（P99－815）,该融合蛋白诱导出小鼠的细胞毒T淋巴细胞（CTL）反应,小鼠的体液免疫反应也较好。这为今后进一步探索慢性乙肝治疗性疫苗奠定了必要的基础。     

丙肝病毒核心蛋白与乙肝病毒核心抗原的融合表达及其性质研究

构建了丙肝病毒核心蛋白 ( 1～ 191)及其N端 ( 1～ 69)和 ( 1～ 40 )与乙肝病毒核心抗原 ( 1～ 14 4 )羧端的融合克隆 ,在大肠杆菌中进行了表达 .其表达产物B14 4C191,B14 4C69和B14 4C40同时具有乙肝核心抗原 (HBc)和丙肝核心蛋白 (HCc)的双重抗原性和免疫原性 .CsCl密度梯度超离心和电镜观察表明 ,融合蛋白能组装成颗粒 .比较等量的融合蛋白的抗原性和免疫原性后发现 ,融合的HCc长度对HBc的抗原性和免疫原性影响不大 .而B14 4C69和B14 4C40比B14 4C191免疫小鼠能产生更高的抗HCc抗体 .利用表达的融合蛋白建立了ELISA法 ,对人血清中抗HBc抗体和抗HCc抗体进行了检测 .     

酿酒酵母表达的rHBcAg颗粒的纯化和鉴定及其透射电镜观察

乙肝核心抗原(HBcAg)蛋白基因(C基因)在酿酒酵母中表达,表达产物经过分离和Sepharose CL-4B柱子的初步纯化.产物经SDS-PAGE和Western blotting鉴定为一分子量约21.5kDa的多肽.再经蔗糖密度梯度超离心和CsCl等密度梯度超离心等过程而被纯化.分管收集的超离心纯化产物经ELISA抗原活性检测和密度分析,可知ELISA反应强度较高的收集管中的颗粒密度主要分布在1.27g/mL和1.40 g/mL两个峰值处.将rHBcAg抗原活性最高的收集管合并,再经TEM观察,发现酵母表达的rHBcAg蛋白(核心蛋白)能自主装配成大小不同的两种核心颗粒,大颗粒直径约为30.1±2.4 nm,小颗粒直径约为21.5±3.3 nm.这表明,酿酒酵母表达的rHBcAg颗粒具有大小不同的二态性,其生物学意义还未明了,需进一步研究和探讨.     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

乙肝病毒核心蛋白及核心颗粒的结构及功能研究进展

乙型肝炎是由嗜肝DNA病毒(Hepatitis B virus,HBV)引起的一种世界范围内的传染性疾病.受HBV慢性感染的人群罹患肝硬化和原发性肝细胞癌(Hepatocellular carcinoma,HCC)的相对危险性至少增加100倍,并最终导致死亡,给人们的健康造成极大的威胁和损害.鉴于此,世界上许多生物医学家早已把他们的研究目标集中到了乙肝病毒,以期弄清楚该病毒的基本结构,致病机理、免疫原性、免疫逃避和生化分子遗传规律等方面的问题.而近来在这些研究中,对乙肝核心抗原(HBcAg)结构、功能及免疫逃避机制的研究又成了引人注目的焦点.本文综合了近年来国内外对乙肝核心抗原(HBcAg)及其核衣壳和核心抗原缺失突变体的研究资料,从以下几个方面对该领域的研究作了较为深入的分析和概述.     

抗原修复方法在乙肝肾免疫组化检测的应用

乙型肝炎病毒相关肾小球肾炎（HBVAssciated Glomerulne phvitis）,简称乙肝性肾炎,临床上常要求对肾穿刺乙肝肾患者的标本做乙肝表面抗原（HBsAg）,乙肝核心抗原（HBcAg）或乙肝e抗原（HBeAg）检测。然而不同的抗原修复方法对肾穿组织石蜡切片HBsAg、     

乙肝核心抗原（HBcAg）作为免疫载体蛋白的研究进展

基因工程疫苗是微生物学免疫研究的热点之一,本文从几方面综述乙肝核心抗原（HBcAg）作为基因工程肽苗免疫载体（Vaccine carrier moety)的研究进展：①HBcAg的结构特征;②HBcAg独特的免疫学性质;③HBcAg作为异源表位递呈系统（Epitope display system)的研究进展。     

呼吸道合胞病毒G蛋白基因片段的克隆及高效表达

呼吸道合胞病毒(RSV)感染遍布全球,并可导致严重的疾病,但目前尚无成功的疫苗问世.为寻求可能用于RSV疫苗研制的重组蛋白抗原,我们在克隆RSV-A全长G蛋白基因的基础上,构建了多种共表达载体蛋白和G蛋白片段的表达载体,并从中筛选出能以可溶形式高效表达抗原蛋白的原核表达体系.通过亲和层析纯化了重组蛋白抗原DsbA-G101,将其免疫Balb/c小鼠后获得了相应的抗血清.经ELISA检测表明DsbA-G101具有良好的免疫原性.基于本研究所构建的系列表达载体,可以比较不同的G蛋白片段免疫原性的强弱及载体蛋白的优劣,从中发现最佳的RSV抗原蛋白.     

铅螯合物人工抗原的制备与鉴定

以双功能螯合剂异硫氰酸苄基乙二胺四乙酸(ITCBE)螯合铅离子,制备得半抗原Pb-ITCBE,然后再分别与载体蛋白KLH或BSA偶联制备得免疫原Pb-ITCBE-KLH与包被抗原Pb-ITCBE-BSA,ITCBE-BSA.用二喹啉甲酸法测3种抗原的浓度,分析半抗原、抗原与载体蛋白的紫外吸收光谱,利用SDS-PAGE对3种抗原的分子量进行鉴定,用三硝基苯磺酸法检测3种抗原中的赖氨酸残基的ε-NH2被半抗原替换的程度,用石墨炉原子分光吸收法检测抗原中铅的含量.研究结果表明,免疫原与包被抗原制备成功,Pb-ITCBE-KLH、Pb-ITCBE-BSA、ITCBE-BSA的浓度依次为6.47± 0.08 mg/ml,6.68± 0.06 mg/ml,5.57± 0.05 mg/ml;抗原与载体蛋白的紫外吸收光谱的特征各不相同;SDS-PAGE的结果显示3种抗原的分子量均不同于各自的载体蛋白;抗原中载体蛋白ε-氨基的替换程度依次为1.86± 0.74 %、55.53± 1.13%、54.19± 1.34%;铅的含量依次为15.64± 0.11 μg/ml,17.33± 0.15 μg/ml,0 μg/ml.     

Comparative immunogenicity of hepatitis B virus core and E antigens

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.     

乙肝病毒核心抗原作为载体用于病毒样颗粒疫苗研究的主要进展

乙肝病毒核心抗原(Hepatitis B virus core antigen,HBcAg)是乙肝病毒的核壳结构蛋白,由183～185个氨基酸组成,大小约21～23kD。HBcAg由于其能自我组装成病毒样颗粒(Virus-like particle,VLP)、高表达、易纯化以及强免疫原性等特点,使其成为一个高效安全且应用广泛的VLP载体,可用于各种病原的疫苗研发。发展至今已有数十种病毒、细菌以及寄生虫的相关基因的抗原表位成功表达在HBcAg VLP颗粒上,成为新型疫苗研发的重要平台。     

Comparative antigenicity and immunogenicity of hepadnavirus core proteins

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.     

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins

Virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. Presentation on the icosahedral hepatitis B virus capsid (HBcAg) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 B cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. Even some complete proteins were successfully inserted but others, e.g. the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi, impaired formation of capsid-like particles (CLPs). This difference can be rationalized by the requirement for the termini of the insert to fit into the predetermined geometry of the two acceptor sites in the carrier. We reasoned that cleavage of one of the two bonds connecting insert and carrier should relieve these constraints, provided the cleaved protein fragments remain competent to support the particle structure. Indeed, HBcAg CLPs containing a recognition site for tobacco etch virus (TEV) protease in the c/e1 loop remained intact after cleavage, as did CLPs carrying a 65-residue peptide insertion. Most importantly, in situ cleavage of a core-OspA fusion protein by coexpressed TEV protease strongly enhanced CLP formation compared with the uncleaved protein. These data attest to the high structural stability of the HBcAg CLP and they significantly widen its applicability as a carrier for heterologous proteins. This approach should be adaptable to any protein-based particle with surface-exposed yet sequence-internal loops.     

Analysis of foreign epitope inserts in HBcAg. Approaches to solving the problem of core particle self-assembly

The hepatitis B virus core antigen (HBcAg) is a promising protein carrier for exposing the epitopes of various human and animal pathogens. HBcAg-based chimeric proteins can be used in creating highly efficient vaccines; however, not all chimeric HBcAg with foreign epitope inserts are capable of assembly into virus-like particles. Using computer programs ProAnalyst, SALIX, and QSARPro, we examined the relationship between the self-assembly capability of chimeric HBcAg and the physicochemical properties of the inserts. The self-assembly was found to be impaired when the inserted peptides contained highly hydrophobic and bulky residues tending to form β-structures; this especially concerned the C-proximal residues in the insert. Recommendations were elaborated for constructing foreign epitopes that would ensure correct self-assembly of chimeric HBcAg particles.     

Mucosal immunogenicity of the hepatitis B core antigen

The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.     

Combinatorial approach to hepadnavirus-like particle vaccine design

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the "preexisting immunity" and "assembly" problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.     

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein

Abstract Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc_3–144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc_1–183) could not be purified or characterised immunologically, although it formed core like particles. HBc_3–144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7–17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc_3–144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.     

通过乙型肝炎病毒核心基因5''''端的修饰使核心抗原在大肠杆菌中高效表达

为了使乙型肝炎病毒核心抗原(HBcAg)在大肠杆菌中获得高效表达,本文首次采用一种新方法对核心基因前区进行改造与修饰,即用限制性内切酶Taq I从核心基因内部5′端切开,去除核心基因起始信号ATG及ATG 5′端上游的全部前核心区(Precore),再化学合成一段既包含核心基因起始信号又具有多种功能的DNA片段。将两者拼接重组到表达载体pUC9上,转化受体菌,成功地获得高效表达HBcAg的菌株。用ELISA法检测,表达滴度为1:80000。表达产量占菌体总蛋白的16％。其菌体裂解液经免疫电镜观察,可见到成堆聚集的典型HBcAg颗粒。抗原单体分子量约为22000道尔顿,双体为44000。与目前国内外所普遍采用的方法比较,本文的方法有许多明显的优点,可用于其它基因改造。