Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.     

Expression of overlapping PreS1 fragment recombinant proteins for the determination of immunogenic domains in HBsAg PreS1 region

The virion of the hepatitis B virus (HBV) is a sphericalparticle of 42-nm diameter whose envelope contains threerelated surface glycoproteins called the large (L), middle(M) and small (S) proteins.All these proteins are expressedfrom one open reading frame using three in-frame startsites [1]. The L protein is the translation product of thewhole open reading frame. The M protein lacks the N-terminal amino acid residue 108–119 of Lprotein (the preS1sequence), and the S protein lacks the N…     

Antibody response to preS1 in hepatitis-B-virus-induced liver disease and after immunization

Antibodies to the preS1-encoded sequence of hepatitis B virus (HBV) envelope were detected by ELISA using a synthetic peptide analogue of preS1 proteins, in different groups of HBV-infected subjects and also in hepatitis B vaccine recipients. Such antibodies were specifically found in only 1 % of HBsAg chronic carriers including patients with cirrhosis and primary liver cancer. Anti-preS1 were detected in patients with acute hepatitis; in 13 % of the HBsAg⁺ sera obtained before recovery and in 37 % of the sera obtained after recovery.Anti-preS1 antibodies were detected in recipients of a plasma-derived vaccine, but not in those receiving a recombinant vaccine. The results indicate that anti-preS1 is an earlier serum marker of HBV clearance than anti-preS2 and anti-S antibodies.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

乙型肝炎病毒表面抗原preS1在大肠杆菌中的表达与鉴定

本文报告利用pWR590质粒为载体,构建了含1ac启动子、β-半乳糖苷酶(1—590)基因、Xa因子的四肽识别位点和HBV preS1、preS2编码序列的表达质粒,并成功地在大肠杆菌中获得稳定表达。融合蛋白经Xa因子消化和高效液相层析,得到了preS1(1—91)纯肽。此肽特异性地与人肝细胞质膜结合,从而为肝细胞上存在preS1受体提供了直接的实验依据,也为分离和鉴定肝细胞上preS1受体打下了良好的基础。     

Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli

The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.     

修饰的含前S1、前S2免疫决定簇乙肝表面抗原融合多肽在毕赤酵母中的共表达

用BamHⅠ和BglⅡ双酶切含SS2嵌合基因片段的重组质粒pAO815-SS2,分离含AOX1启动子区、SS2嵌合基因和AOX1转录终止序列的表达盒。将分离的BamHⅠ-BglⅡ表达盒与经BamHⅠ线性化的pAO815-SS1质粒连接,转化大肠杆菌Top10F′,提取质粒,用BamHⅠ和BglⅡ双酶切分析重组质粒并筛选正向插入SS2表达盒的重组子。将该重组质粒用电转化法导入PichiapastorisGS115细胞,经表型筛选、小试表达和产物鉴定,构建了重组表达菌株GS115-SS1S2。重组菌株经甲醇诱导后制备抗原粗提液,进一步进行ELISA和Westernblot鉴定。ELISA结果显示,产物同时具有S、前S1和前S2抗原性。Westernblot分析进一步表明,表达产物既能与S抗体结合,也能与前S1和前S2抗体结合。工程菌经高密度发酵,表达量达到300~600mg/L。抗原经纯化后进行电镜观察,形成直径20~35nm的球形颗粒。纯化抗原经SDS-PAGE分析,SS1和SS2多肽仍然保持完整,基本无降解。     

Efficient expression,purification and characterization of hepatitis B virus preS1 protein from Escherichia coli

Summary The complete (encoding 119 aminon acids, aa) or partial (encoding the N-terminal 90 aa) preS1 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathione-S-transferase (GST) gene and expressed at 37 °C under the control of the inducible tac promoter in E. coli. The results showed that the fusion protein with the full length of preS1 was moderately expressed, about 10% of total cellular proteins, while the protein with the partial preS1 was highly expressed, about 33% of total cellular proteins but the half was degraded into the protein with about N-terminal 60 aa of preS1. Accordingly, GST fusion protein containing the N-terminal 56 aa of the preS1, which still encodes B-and T-cell epitopes and a hepatocyte receptor binding site, was expressed under the same induction conditions and was shown to be highly and stably expressed, about 37% of total cellular proteins. The fusion protein with the full length or N-terminal 56 aa of preS1 and the peptides were simply and successfully purified by affinity chromatography and were demonstrated to exhibit the antigenicity and immunogenicity of the preS1 antigen.     

Real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance

The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

Bacterial expression,purification, and in vitro N-myristoylation of fusion hepatitis B virus preS1 with the native-type N-terminus

Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.     

乙型肝炎病毒S-preS1-C融合基因在酵母中的表达及抗原性分析

以HBV全基因组质粒为模板,扩增出目的区段:S(1-222AA),preS1(10-50AA)和C(2-30AA),以酶切-PCR的方法进行连接,克隆入表达载体pPIC3.5k,电穿孔转化后利用G418加压筛选出多拷贝整合菌株,诱导表达并对表达产物进行检测。结果显示融合后的基因为ss1c,长度约为906bp。在酵母中成功地进行了表达,表达产物(SSIC)的大小约为31kDa,并形成直径约为22nm的病毒样颗粒。表达产物与HBs抗体、preS1抗体和HBc抗体均有特异反应。为进一步研究治疗性疫苗提供了重要的基础。     

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Phages with high affinity to the S protein obtained fromrice stripe virus (RSV) were enriched fromphage-displayed random 12-mer peptide library after threerounds of biopanning. 9 different peptides from theenriched library were selected by ELISA. Circulardichroism (CD) spectra of the GST-S fusion protein withbinding phages and non-binding phages showed thatstructure of the S protein was changed after it bound toeach of these 9 selected 12-mer peptides, which suggestedthat these peptides might disrupt the function of Sprotein. Thus, those peptides might be used to developplant resistance and disrupt virus transmission. 3 of the12-mer peptide genes were fused with the GST gene in pGEX3X. The fusion proteins were also obtained using E.coli expression system and purified.     

乙型肝炎病毒表面抗原前S区和谷胱甘肽巯基转移酶融合基因在大肠杆菌中的表达

我们构建了谷胱甘肽巯基转移酶（ＧＳＴ）和完整的或部分缺失的乙型肝炎病毒表面抗原前Ｓ区的融合基因,并在大肠杆菌中进行了表达。融合蛋白的产量随着前Ｓ区长度的增加而迅速降低,而且融合蛋白的前Ｓ区有严重的降解,主要降解位点在ｐｒｅＳ１区的ａ．ａ．７５和ｐｒｅＳ２区的ａ．ａ．１３０和ａ．ａ．１６５左右。利用蛋白降解酶系缺陷型菌株进行的研究表明,这种降解酶存在于多个大肠杆菌株中而且和大肠杆菌中的两个主要的蛋白降解酶系Ｌｏｎ和ｈｔｐＲ无关。具有重要生物学功能的前Ｓ区肽段（ｐｒｅＳ１ａ．ａ．１－６５）因含有阻止分泌的滞留顺序而无法在哺乳动物细胞和酵母中大量表达,但滞留顺序的存在并没有影响含有这一肽段的融合蛋白在大肠杆菌中的表达和产物的纯化。ＧＳＴ融合表达系统产量高,纯化快速简便。用这一方法大量表达并得到的这一肽段不仅是研究乙型肝炎病毒的分子生物学的重要材料,而且可以作为新一代乙型肝炎疫苗的主要组成成分。     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

An anti-viral peptide derived from the preS1 surface protein of hepatitis B virus

The preS1 surface protein of the hepatitis B virus (HBV) is a key factor involved in initial viral entry into hepatocytes. It has been long postulated that an anti-HBV effect should be achievable using peptide fragments of the preS1. Recent reports demonstrated that several preS1-derived lipo-peptides in genotype D HBV exhibit nano to picomolar inhibitory activity against HBV infection. In this study, an acylated analog of a preS1 fragment, a 21-residue lipo-peptide (named 7524 BVS7) with a sequence of palmitoyl-GMGTNLSVPNPLGFFPDHQLDC-NH2, from genotype C HBV was produced base upon a previous structural study and was shown potently inhibits HBV infection with an IC(50) of approximately 20 nM.     

HepG2细胞膜上乙型肝炎病毒前S1蛋白（preS1）结合蛋白的研究

采用pull down技术研究preS1在HepG2细胞膜上的结合蛋白。以原核表达的GST-preS1融合蛋白为探针蛋白,与生物素标记的HepG2细胞裂解液进行pull down试验分离与preS1结合的膜蛋白。Western blot结果显示HepG2细胞膜上有一大小约110kDa蛋白（p110）与preS1结合。通过对比实验证明该蛋白具有较好的组织特异性和种属特异性。研究结果显示该蛋白是HepG2细胞膜上与preS1结合的蛋白,可能与HBV的早期感染过程有关。     

Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1

Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.     

肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析

HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 ,评估其在临床肿瘤诊断中的作用