大肠杆菌合成的HBcAg转化为HBeAg的研究及其应用

37℃用1％β-巯基乙醇处理大肠杆菌HBcAg2小时,可使其全部转化成HBeAg。以常规ELISA方法不能检出HBcAg活性。该制品能被抗-HBe阳性血清特异中和。4℃以液体形式存放稳定,-20℃经过冻融活性下降。HBcAg转化成HBeAg后聚丙烯酰胺凝胶电泳行为有所改变。分别用转化的大肠杆菌HBeAg和血浆HBeAg作为酶联反应的诊断试剂,检测35份HBsAg阳性乙肝病人血清标本的抗-HBe,符合率为91.4％。同用Abbott-HBe(RIA)试剂盒测定的17份HBsAg阳性血清标本的试验结果比较,符合率为100％。说明转化的大肠杆菌HBeAg可代替血浆HBeAg作为检测抗-HBe的诊断试剂应用。     

重组乙型肝炎病毒e抗原的研究进展

乙型肝炎e抗原（HBeAg）是由HBV DNA C基因区编码的一种非颗粒性的分泌型核壳蛋白,临床上将其作为判断HBV活动性复制的指标之一。HBeAg是重要的生物原材料,广泛用于HBV感染者血清学检测的相关诊断用品的制备。重组HBeAg的表达和纯化技术较为成熟,已经在各种表达系统中成功地表达了目的蛋白。而影响其表达的关键因素包括前C区重要位点变异、载体选取以及RNA干扰（RNAi）的影响作用等。因此,提高重组HBeAg的表达量和纯度,避免与HBcAg交叉反应才能满足相关诊断制品的要求。高质量重组乙肝e抗原的获取,能为改善相关诊断制品奠定物质基础,为开发新型HBV治疗和预防性疫苗提供了依据,同时也为HBeAb检测方法学的优化提供可能。     

大肠杆菌合成的乙型肝炎病毒核心抗原的研究Ⅱ．HBcAg的提纯

本文报道HBcAg基因工程菌MM206株的粗提物通过DEAE柱后,基因表达产物HBcAg与核酸及大部分细菌蛋白分开后,再经凝胶过滤和亲和层析作进一步提纯。经DEAE层析和亲和层析获得的纯制品聚丙烯酰胺凝胶电泳图谱显示一主带和两小带。从sepharose 4B柱和亲和层析柱洗出的HBcAg,ELIsA检测证明其比活性分别比粗制品高40倍和100倍,以纯制的HBcAg注射家兔,在6只接受免疫的动物中均产生有抗一HBc,能与从人肝提取的HBcAg产生特异的免疫反应,这表明大肠杆菌合成的HBcAg在动物体内具有生物学活性。     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

用DNA重组技术获得乙型肝炎病毒e抗原及其在临床检测中的应用

HBeAg和抗－Hbe是乙型肝炎的经典标志物。HBeAg基因与HBcAg基因在DNA c区上相重叠。为要获得重组DNA衍生的HBeAg,本文从adw2亚型HBv 3．2 kb全长DNA上切取带有HBc基因的BamHI-EcoRI片段,用Bal3l和Hpa I酶处理产生一系列长度不同的片段,与载体pUR222和pUC 18构成不同的重组质粒,转化到大肠杆菌。通过平板筛选、质粒酶切图谱和抗原抗体反应等筛到高效表达HBeAg的转化株YG30l和YG302,中和试验证明了重组HBeAg的血清学特异性。用凝胶过滤、离子交换层析和亲和层析对其进行提纯并用聚丙烯酰胺凝胶电泳结合western印迹法进行纯度鉴定、含量分析和分子量测定。重组HBeAg制品具有高活性、稳定和安全等特点,实验证明它可以代替血源HBeAg制成酶联药盒用于乙肝诊断检测。     

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。     

重组风疹病毒抗原的分离纯化

为了制备临床诊断用的风疹病毒抗原,建立了亲合色谱分离纯化的方法.风疹抗原以可溶性形式在大肠杆菌工程菌中获得高效表达,用GST亲合色谱在不变性的条件下直接从细菌裂解液中分离纯化.纯化的目的蛋白电泳为单一条带,EUISA试验表明,重组抗原与风疹病毒IgM阳性血清能特异反应,而与IgM阴性血清不反应,表明重组蛋白具有良好的抗原性,能满足临床检验要求.     

风疹病毒多表位诊断抗原的制备与评价

原核表达与层析纯化获取风疹病毒(Rubella Virus,RV)多表位诊断抗原,并初步评价其抗原性。将RV衣壳蛋白C aa 3~29、aa 96~123以及包膜糖蛋白E1aa208~247三个主要免疫显性表位串联,密码子优化后全基因合成;利用基因重组技术将多表位串联片段插入带有GST标签的表达质粒中,在大肠杆菌中进行表达,并利用亲和层析和离子交换层析纯化目的蛋白;利用Western Blot(WB)技术对目的蛋白抗原性进行鉴定,并建立RV-IgM抗体检测ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。获取高度均质的RV多表位诊断抗原,WB实验表明目的蛋白不但可以被anti-GST单克隆抗体识别,还可以被anti-RV多克隆抗体、RV-IgM阳性血清相应抗体所识别。分别对48份RV急性感染病例的阳性血清、阴性血清和健康人血清进行检测,发现一致性优异。原核表达与层析纯化可以获取抗原性良好的RV多表位诊断抗原,偶联HRP后可以作为检测抗原应用于RV-IgM ELISA血清学检测中。     

利用基因工程表达核心抗原转化的E抗原制备抗HBeAg单克隆抗体

采用两种变性剂将大肠杆菌基因工程高效表达的乙型肝炎病毒核心抗原(HBcAg)转化成为E抗原(HBeAg),其ELISA滴度达1:1000以上,而核心抗原滴度为零。用此抗原免疫小鼠,一次融合即获十四株抗乙型肝炎病毒E抗原的单克隆抗体。细胞培养上清抗体滴度为1:10-1:1000以上,腹水滴度为1:1000-1:100,000以上。十三株IgG、一株IgM。其中十株为抗HBeAg(b)决定簇,四株抗(a)决定簇。它们的敏感性及特异性与日本用血清E抗原制备的单克隆抗体效果完全一致。这是国内外首次应用基因工程表达核心抗原转化E抗原成功 地建立分泌抗HBeAg单克隆抗体杂交瘤细胞株。     

HBcAg表达菌株的筛选、初步鉴定与DNA顺序分析

为提高HBcAg(乙型肝炎核心抗原)在大肠杆菌体内表达水平,将有HBcAg的基因片段用核酸外切酶从两端消化,插入载体质粒pUR222的EcoRI酶切位点,转化Ecoli BMH71—18,用菌落原位酶联免疫法由275个转化子筛选到7株阳性克隆,用ELISA比较了它们的表达水平,表达水平最高者为M2066菌株,P/N=2.0时菌体裂解液的稀泽度为1:33000’比表达最低者M2098高8,000倍,比第一代菌株M206高15,000倍,DNA顺序分析结果表明与mRNA起始密码上游的发卡结构去除有关。用SDS-PAGE和聚乙烯簿膜复印法检测菌体裂解液中HBcAg的分子量为21000,42000及63000,呈单体和聚合物形式存在,比由病人肝脏和血液中提取的HBcAg(19000)分子量大,为融合蛋白。经琼脂免疫双扩散与ELISA阻断试验未发现与β半乳糖苷酶有免疫学交叉反应。用ELIDA法,M2066-HBcAg与肝-HBcAg同时检测40份血清标本的抗-HBc,二者符合率95％。     

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.     

大肠杆菌表达的乙型肝炎病毒核心抗原的某些理化特性

1979年Pasek等用大肠杆菌表达乙型肝炎病毒核心抗原(HBcAg)成功以来,国内外也有类似的报道,但对其理化特性的报道不多。为了更有效地保存和应用此抗原,本文研究了从表达HBcAg的大肠杆菌菌液中纯化抗原的方法,以及抗原的某些特性。 HBcAg是由pHBV-1的C基因与pTL载体重组后在大肠杆菌BMH71-18内表达的,     

DNA-binding activity of hepatitis B e antigen polypeptide lacking the protaminelike sequence of nucleocapsid protein of human hepatitis B virus.

The characteristics of binding of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) polypeptides to hepatitis B virus (HBV) DNA were analyzed. HBcAg polypeptide from recombinant HBV core particles and HBeAg polypeptide from partially purified serum HBeAg were prepared and verified to have molecular weights of 21,500 (P21.5) and of 17,000 (P17) and 18,000 (P18), respectively, by immunoblot analysis. By reaction of these proteins on a nitrocellulose membrane with cloned 32P-HBV DNA, it was revealed that the HBeAg polypeptide, which lacks the C-terminal 34 amino acids of P21.5, as well as the HBcAg polypeptide, bound to the DNA. The secondary structures of nucleocapsid proteins of HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus were predicted by the Garnier algorithm. Amino acid sequences which, in addition to those of the C-terminal regions, may contribute to binding were proposed to be the 21-amino-acid residues located at amino acids 100 to 120 of the nucleocapsid proteins of these hepadnaviruses.     

含不同长度前C序列的乙型肝炎病毒核心抗原基因在重组痘苗病毒中的表达

核酸序列分析表明,乙型肝炎(乙肝)病毒核心抗原基因(C基因)编码区内存在两个ATG。目前认为第二个ATG为乙肝病毒C抗原的起始密码子,两个ATG之间的序列称为前C序列,共87bp。Uy、Rutter、Roossinck及景新等的研究表明,含前C序列时,C基因在大肠杆菌中的表达是形成具有HBeAg活性的P25~e膜蛋白,在哺乳动物细胞中的表达则是形成分泌性的HBeAg;不含前C序列时,在大肠杆菌和哺乳动物细胞中均形成P21~e     

Negative HBcAg in immunohistochemistry assay of liver biopsy is a predictive factor for the treatment of patients with nucleos(t)ide analogue therapy

The hepatitis B core antigen (HBcAg) is an important target for antiviral response in chronic hepatitis B (CHB) patients. However, the correlation between HBcAg in the hepatocyte nucleus and nucleos(t)ide analogue (NA) therapeutic response is unclear. We sought to evaluate the role of HBcAg by analysing liver biopsies for viral response in NA‐naïve hepatitis B e antigen (HBeAg) positive (+) CHB patients via immunohistochemistry (IHC). A total of 48 HBcAg‐negative (?) patients and 48 HBcAg (+) patients with matching baseline characteristics were retrospectively analysed for up to 288 weeks. Virological response (VR) rates of patients in the HBcAg (?) group were significantly higher at week 48 and 96 than the HBcAg (+) group (77.1% versus 45.8% at week 48, respectively, P = 0.002 and 95.3% versus 83.3% at week 96, respectively, P = 0.045). The serological negative conversion rate of HBeAg was significantly higher in the HBcAg (?) than in the HBcAg (+) group from week 96 to 288 (35.4 % versus 14.6% at week 96, respectively, P = 0.018; 60.4% versus 14.6%, respectively, P < 0.001 at week 144; 72.9% versus 35.4%, respectively, P < 0.001 at week 288). The cumulative frequencies of VR and lack of HBeAg were higher in the HBcAg (?) group (both P < 0.05). Binary logistic regression analysis showed that HBcAg (?) was the predictor for the lack of HBeAg (OR 4.482, 95% CI: 1.58–12.68). In summary, the absence of HBcAg in the hepatocyte nucleus could be an independent predictor for HBeAg seroconversion rates during NA‐naïve treatment in HBeAg (+) CHB patients.     

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.     

Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection.

Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti-HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection.     

The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype.

Secretion of the hepatitis B virus (HBV) e antigen (HBeAg) has been conserved throughout the evolution of hepadnaviruses. However, the function of this secreted form of the viral nucleoprotein remains enigmatic. It has been suggested that HBeAg functions as an immunomodulator. We therefore examined the possibility that the two structural forms of the viral nucleoprotein, the particulate HBV core (HBcAg) and the nonparticulate HBeAg, may preferentially elicit different T helper (Th) cell subsets. For this purpose, mice were immunized with recombinant HBcAg and HBeAg in the presence and absence of adjuvants, and the immunoglobulin G (IgG) isotype profiles of anti-HBc and anti-HBe antibodies were determined. Second, in vitro cytokine production by HBcAg- and HBeAg-primed Th cells was measured. The immunogenicity of HBcAg, in contrast to that of HBeAg, did not require the use of adjuvants. Furthermore, HBcAg elicited primarily IgG2a and IgG2b anti-HBc antibodies, with a low level of IgG3, and no IgG1 anti-HBc antibodies. In contrast, the anti-HBe antibody response was dominated by the IgG1 isotype; low levels of IgG2a or IgG2b anti-HBe antibodies and no IgG3 anti-HBe antibodies were produced. Cytokine production by HBcAg- and HBeAg-primed Th cells was consistent with the IgG isotype profiles. HBcAg-primed Th cells efficiently produced interleukin-2 (IL-2) and gamma interferon (IFN-gamma) and low levels of IL-4. Conversely, efficient IL-4 production and lesser amounts of IFN-gamma were elicited by HBeAg immunization. The results indicate that HBcAg preferentially, but not exclusively, elicits Th1-like cells and that HBeAg preferentially, but not exclusively, elicits Th0 or Th2-like cells. Because HBcAg and the HBeAg are cross-reactive in terms of Th cell recognition, these findings demonstrate that Th cells with the same specificity can develop into different Th subsets based on the structural form of the immunogen. These results may have relevance to chronic HBV infection. Circulating HBeAg may downregulate antiviral clearance mechanisms by virtue of eliciting anti-inflammatory Th2-like cytokine production. Last, the influence of antigen structure on Th cell phenotype was not absolute and could be modulated by in vivo cytokine treatment. For example, IFN-alpha treatment inhibited HBeAg-specific Th2-mediated antibody production and altered the IgG anti-HBe isotype profile toward the Th1 phenotype.