TT病毒重组蛋白单克隆抗体的制备

采用杂交瘤技术,获得了4株稳定分泌抗TTV重组蛋白的单克隆抗体杂交瘤细胞株,1株属IgG2bλ链、1株属IgG1κ链、2株属IgG2aκ链。4株杂交瘤细胞培养上清液效价为1：80-1：1280,腹水效价为1：32万-1：160万。     

降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。     

Synthesis and immunochemical properties of peptides corresponding to sequences 59-72 and 25-36 of human interleukin-2

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.     

Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

Formation of a uniquely stable type I interferon receptor complex by interferon beta is dependent upon particular interactions between interferon beta and its receptor and independent of tyrosine phosphorylation

Human type I interferons (IFN) require two receptor chains, IFNAR1 and IFNAR2c for high affinity (pM) binding and biological activity. Our previous studies have shown that the ligand dependent assembly of the type I IFN receptor chains is not identical for all type I IFNs. IFNbeta appears unique in its ability to assemble a stable complex of receptor chains, as demonstrated by the observation that IFNAR2c co-immunoprecipitates with IFNAR1 when cells are stimulated with IFNbeta but not with IFNalpha. The characteristics of such a receptor complex are not well defined nor is it understood if differential signaling events can be mediated by variations in receptor assembly. To further characterize the factors required for formation of such a stable receptor complex we demonstrate using IFN stimulated Daudi cells that (1) IFNAR2c co-immunoprecipitates with IFNAR1 even when tyrosine phosphorylation of receptor chains is blocked with staurosporine, and (2) IFNbeta1b but not IFNalpha2, is present in the immunoprecipitated receptor complex. These results demonstrate that the unique IFNbeta induced assembly of type I IFN receptor chains is independent of receptor tyrosine phosphorylation and the recruitment of additional proteins to the receptor by such events. Furthermore, the presence of IFNbeta1b in the immunoprecipitated IFN receptor complex suggests that IFNbeta interacts and binds differently to the receptor than IFNalpha2. These results suggest that the specific assembly of type I IFN receptor chains is ligand dependent and may represent an early event which leads to the differential biological responses observed among type I IFNs.     

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Background

Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results

We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion

These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.     

Determination and validation of off-target activities of anti-CD44 variant 6 antibodies using protein biochips and tissue microarrays

We describe a novel procedure for determination and validation of off-target activities of anti-cluster designation antigen identity 44 (CD44) variant 6 recombinant antibodies by combining two complementary technology platforms; namely UNIchip AV-400, containing a printed serial dilution of CD44 variant 6 and approximately 400 different human proteins, and TISSOMICS, enabling human tissue microarray analysis in high-throughput mode. We have analyzed the performance of two human monoclonal recombinant antibodies directed against CD44 variant 6 protein, BMS 116 and BMS 125, in a blinded study. The antibodies exhibit a clear differentiation with regard to their binding profiles in the two systems. BMS 116 shows a low degree of specificity in the normal human Food and Drug Administration (FDA) tissue panel, which was confirmed by binding to more than 206 proteins on the protein biochip. In contrast, BMS 125 gives a highly selective membranous staining on selected human epithelial tissue components with no off-target activities observed on the protein biochip. Additionally, antibody BMS 125 shows a higher sensitivity to its antigen CD44 variant 6 than antibody BMS 116 as determined by the protein biochip.     

Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (tiantan)-based HBV vaccine

Background

A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/Principal Findings

To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions

The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.     

人IL-2/IFNα2b融合基因在肝癌细胞中靶向表达

 根据细胞因子之间协同作用的特点,采用重组 Ｄ Ｎ Ａ 技术设计并构建了人 Ｉ Ｌ ２ 与 Ｉ Ｆ Ｎα融合基因,并用肝癌组织特异的 Ａ Ｆ Ｐ增强子／ Ａ Ｌ Ｂ启动子调控融合基因在肝癌细胞中的靶向表达．实验结果表明,克隆的 Ｅ Ａ Ｆ Ｐ Ｐ Ａ Ｌ Ｂ联合转录调控序列能调控细胞因子基因在 Ａ Ｆ Ｐ阳性人肝癌细胞中靶向表达, Ｉ Ｌ ２／ Ｉ Ｆ Ｎα２ｂ 融合基因的表达水平与感染肝癌细胞的 Ａ Ｆ Ｐ表达水平呈正相关性．实验证明表达的融合蛋白具有 Ｉ Ｌ ２ 和 Ｉ Ｆ Ｎ 两种生物学活性的细胞因子．这可能为肝癌基因治疗开辟新途径．     

Profiles of IgG antibodies to nucleocapsid and spike proteins of the SARS-associated coronavirus in SARS patients

To evaluate humoral immunity against the SARS-associated coronavirus (SARS-CoV), we studied the profiles of IgG antibodies to the nucleocapsid (N) and spike (S) proteins of SARS-CoV. Serum specimens from 10 SARS patients were analyzed by Western blotting and an enzyme-linked immunosorbent assay (ELISA) using purified recombinant N and truncated S (S1, S2, and S3) proteins as antigens. Western blotting results demonstrated that 100% of the SARS patients tested positive for N protein-specific antibodies, 50% for S1 protein-specific antibodies, 30% for S2 protein-specific antibodies, and 70% for S3 protein-specific antibodies. The ELISA results, which showed positive rates of IgG reactivity against recombinant proteins N, S1, S2, and S3, were, respectively, 28.57, 14.29, 14.29, and 14.29% at week 1, 77.78, 55.56, 44.44, and 66.67% at week 2, 100, 75, 75, and 87.5% at week 3, and 100, 77.78, 77.78, and 88.89% after 3 weeks. The average titers of IgG against recombinant proteins N, S1, S2, and S3 were, respectively, 691, 56, 38, and 84 after 3 weeks. These results suggest that the recombinant proteins N and S3 are potentially useful antigens for a serological diagnosis of SARS. In consideration of possible cross-reactivity among N proteins of SARS-CoV and other coronaviruses, immunoassays using recombinant N protein in combination with S3 as antigens might improve the specificity of SARS diagnoses.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2.

Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling.     

Purification of therapeutic proteins mediated by in vivo polyester immobilized sortase

Objectives

To overcome laborious and costly procedures often associated with therapeutic protein production and purification, in vivo polyester immobilized sortase is explored for the production of human tumor necrosis factor alpha (TNFα) and human interferon alpha 2b (IFNα2b) by Escherichia coli.

Results

Hybrid genes encoding PhaC-Sortase-TNFα or PhaC-Sortase-IFNα2b fusions (with a LPETG recognition signal immediately before TNFα or IFNα2b), mediated intracellular production of polyester (polyhydroxyalkanoate, PHA) beads in Escherichia coli. Upon isolation of respective PHA beads, pure soluble TNFα or IFNα2b was released by activating sortase via addition of CaCl₂ and triglycine. TNFα and IFNα2b each were recognized by corresponding conformational antibodies in an ELISA assay.

Conclusions

In vivo polyester immobilized sortase could be exploited for production and purification of high-value therapeutic proteins without laborious and costly downstream processing.

     

Aspects of innate immunity in Sjögren's syndrome

Previously, a dominant role of the adaptive immune system in the pathogenesis of Sj?gren's syndrome was suspected. Recent advances, however, have revealed a major role of the type I IFN pathway, documented by an increased circulating type I IFN activity and an IFN 'signature' in peripheral blood mononuclear cells and minor salivary gland biopsies from the patients. Polymorphisms in the genes IRF5 and STAT4 leading to increased IFN activation are associated with disease susceptibility. In the pathogenesis of Sj?gren's syndrome, the activation of salivary gland epithelial cells appears to be the initial event. Once intrinsically activated, they express costimulatory and Toll-like receptors (TLRs) and MHC class I and II molecules, can present autoantigens and produce proinflammatory cytokines. The subsequent activation of plasmacytoid dendritic cells induces the production of high levels of proinflammatory cytokines in individuals with the risk alleles of the susceptibility genes IRF5 and STAT4. Under the influence of the high IFN concentration in the glands and through TLR ligation, B-cell activating factor is produced by epithelial cells and, together with autoantigen presentation on salivary gland epithelial cells, stimulates the adaptive immune system. In view of the central role of IFNalpha in at least the initiation of the pathogenesis of Sj?gren's syndrome, blockade of this cytokine may be a rational therapeutic approach.     

Human interleukin 1 is a cytocidal factor for several tumor cell lines

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.     

人心肌型脂肪酸结合蛋白单克隆抗体的制备及定量检测试剂盒的研制

目的：利用抗心肌型脂肪酸结合蛋白单抗,研制定量检测心肌型脂肪酸结合蛋白（ H-FABP ）的ELISA试剂盒。方法使用基因重组H-FABP免疫小鼠,以杂交瘤技术制备特异性抗H-FABP单抗,用这些单抗研制定量检测H-FABP的ELISA 试剂盒。结果筛选获得2株稳定分泌抗H-FABP单抗的杂交瘤细胞株,研制了定量检测H-FABP的ELISA试剂盒,灵敏度达到0．2 ng/mL,线性范围0．4～25 ng/mL,r2＝0．9967,回收率在97．2％～104．5％,精密度的变异系数（CV）≤6．72％;应用此试剂盒检测健康人血浆H-FABP,含量为1．87～8．50 ng/mL。结论所研制的ELISA试剂盒有较好的灵敏度及特异性,可用于人血浆中H-FABP含量的检测。