猪传染性胃肠炎病毒重组核蛋白免疫原性的研究

猪传染性胃肠炎病毒（TGEV）核蛋白（NP）基因是高度保守序列．其编码的NP是病毒粒子的主要组成成分。N基因在PCR分离后，亚克隆到PQE30载体上的组氨酸亲合标志的N端，产生与组氨酸融合的重组融合N蛋白，经Ni-NTA金属亲合层析纯化后免疫家兔，产生了多克隆抗体，此抗体与相应的抗原反应所产生的特异性条带和此抗原与抗NP单克隆抗体反应所产生的特异性条带一致。证明重组融合N蛋白具有良好的免疫原性和反应原性。     

Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.     

A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.     

Two Akabane virus glycoprotein Gc domains induce neutralizing antibodies in mice

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1–97 and 189–397 (Gc_1–97 and Gc_189–397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.     

Epitopes and nuclear localization analyses of canine distemper virus nucleocapsid protein by expression of its deletion mutants.

A series of nucleocapsid protein (NP)-deleted genes of the Onderstepoort strain was constructed in order to locate antigenic regions of the NP of canine distemper virus. The expression of proteins from 5'-deleted NP genes was examined in COS-7 cells by indirect immunofluorescence assay using three monoclonal antibodies (MAbs), c-5, f-5 and h-6, and a rabbit serum against NP. These MAbs reacted with two regions of NP. Amino acid residues from 1 to 80, and 337-358, were necessary and sufficient for formation of the epitopes identified by MAbs f-5 and h-6, and c-5, respectively. The proteins translated from intact or 3'-deleted genes were found to be localized in the nuclei of COS-7 cells, whereas the proteins from the 5'-deleted genes were mainly detected in the cytoplasm. These results suggested that 80 amino acid residues at the N-terminus are required for transportation of NP into the nucleus.     

Localization of the antigenic sites of newcastle disease virus nucleocapsid using a panel of monoclonal antibodies

A panel of six monoclonal antibodies (mAbs) against the nucleocapsid (NP) protein of Newcastle disease virus (NDV) was produced by immunization of Balb/c mice with purified recombinant NP protein. Western Blot analysis showed that all the mAbs recognized linearized NP epitopes. Three different NP antigenic sites were identified using deleted truncated NP mutants purified from Escherichia coli. One of the antigenic sites was located at the C-terminal end (residues 441 to 489) of the NP protein. Two other antigenic sites were located within the N-terminal end (residues 26-121 and 122-375). This study demonstrates that the N- and C-terminal ends of the NP proteins are responsible in eliciting immune response, thus it is most likely that these ends are exposed on the NP.     

H9N2亚型禽流感病毒NP蛋白原核表达及多克隆抗体制备

【目的】对华南地区分离到的1株H9N2亚型禽流感病毒(Avian influenza virus, AIV)流行株NP蛋白进行原核表达,制备H9N2亚型AIV NP蛋白多克隆抗体。【方法】将1株H9N2亚型AIV的NP基因克隆至pET-32a(+)原核表达载体,构建NP蛋白原核表达质粒。将重组质粒同时转化大肠杆菌BL21(DE3)和Rosetta(DE3)感受态细胞,经IPTG诱导表达重组蛋白。通过考马斯亮蓝染色和Western blotting分析并比较NP蛋白在两种表达菌中的表达量,进一步优化诱导剂浓度、诱导时间及诱导温度等条件,提高蛋白的表达效率。采用镍柱亲和层析法对NP蛋白进行纯化,用BCA法测定蛋白浓度。以纯化的NP蛋白免疫新西兰大白兔获得多克隆抗体,通过Western blotting和间接免疫荧光对所制备的多克隆抗体进行鉴定,通过间接ELISA测定抗体效价。【结果】试验成功构建pET-32a-H9N2-NP原核表达质粒并表达重组NP蛋白。考马斯亮蓝染色和Western blotting结果均表明,重组NP蛋白在大肠杆菌Rosetta(DE3)感受态细胞中表达水平远高于大肠...     

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.     

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (PEDV) antibodies

An enzyme-linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of porcine epidemic diarrhea (PEDV) infection. The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein was migrated at 48 kDa and reacted with six histidine tag specific monoclonal antibody by immunoblotting. Recombinant N protein ELISA (rnELISA) demonstrated 98.7% specificities among (80) PEDV-free individuals, and 98% sensitivity ranging among (103) clinical samples with PEDV. On testing 884 field samples, an overall agreement of 88.3% was generated between the SN and rnELISA. Taken together, these results indicated that nucleocapsid protein may be a useful antigen for the sera-diagnosis of PEDV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies to PEDV.     

禽流感病毒NP蛋白单克隆抗体的制备与鉴定

为获得禽流感病毒(Avian influenza virus, AIV)NP蛋白的单克隆抗体,将构建的重组表达质粒pET-30a-NP转化BL21细胞,经IPTG诱导表达、纯化后作为免疫原,免疫8周龄Balb/c雌性小鼠,并按常规方法制备杂交瘤细胞。通过ELISA方法、Western-blot方法进行筛选,获得3株杂交瘤细胞株,命名为3A6、2H12、5F7,并进行了培养特性、分泌抗体活性、分泌抗体亚类的鉴定。结果显示：3株细胞株连续传10代均稳定分泌单克隆抗体,分泌的单克隆抗体亚型均为IgG2b,轻链类型均为Kappa。制备并纯化了以上3株单克隆抗体,浓度分别为2.9、2.5、2.8 mg/mL,纯度不低于90%。West-blot检测,单抗与H7血凝抑制试验抗原、H5血凝抑制试验抗原、H9N2病毒能发生特异性反应,说明单抗具有广谱性,且与IBDV、REV、IBV、MDV、ALV、AE、ILT、NDV、EDSV等均无特异性条带出现,说明特异性良好。本研究制备的3株针对禽流感NP蛋白的单抗,具有较好的特异性、保守性和广谱性,为下一步开展AIV诊断试剂如IFA检测试剂盒、ELISA检测试...     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Nucleotide sequence and vaccinia expression of the nucleoprotein of a highly virulent, neurotropic strain of Newcastle disease virus

The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB.     

小反刍兽疫病毒M基因截短表达及鉴定

目的将小反刍兽疫病毒M蛋白基因截短表达后用于特异性单抗制备及临床抗体水平检测。方法:用在线分析软件BepiPred分析小反刍兽疫病毒M蛋白潜在的B细胞线性表位,以本实验室构建的pCR2.1T-PPRV M质粒为模板,扩增三段截短的M基因,纯化后的PCR产物分别与克隆载体pCR2.1T连接,筛选出的阳性重组质粒经双酶切后,分别与表达载体pET-32a(+)及pGEX-6p-1连接,再将鉴定为阳性的重组质粒转化入E.coli BL21(DE3)菌株诱导表达,并用SDS-PAGE及Western blot验证。结果 PCR产物电泳,得到与预期大小相符的特异性片段。对连接克隆载体及表达载体的重组质粒双酶切后,均出现与预期一致的片段,DNA测序表明,插入片段序列与小反刍兽疫Nigeria75/1株M蛋白基因完全一致。重组蛋白经SDS-PAGE及Western blot鉴定,证明所构建的重组蛋白均获得高效表达,并均具有良好的反应原性。结论成功表达了小反刍兽疫截短M基因的蛋白,为制备特异性单抗及小反刍兽疫抗体检测奠定了一定基础。     

Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera

Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.     

Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts

OBJECTIVE: To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. ANIMALS: 18 adult horses. PROCEDURES: 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. RESULTS: Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. CONCLUSIONS AND CLINICAL RELEVANCE: S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.     

猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及其单克隆抗体的制备

试验通过RT-PCR法扩增出猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pet-30a中,转入到BL21感受态细胞,经IPTG诱导表达,使用SDS-PAGE和Western blotting分析表达产物的特性。利用Ni柱纯化该重组蛋白,采用NC膜皮下包埋法免疫Balb/C小鼠,取免疫后的脾细胞与SP2/0细胞进行融合后筛选杂交瘤细胞株,检测杂交瘤细胞株的特性并制备单克隆抗体。SDS-PAGE和Western blotting结果表明,该重组蛋白表达正确,约为20 ku,能被抗PRRSV的阳性血清特异性识别。超声后用Ni柱纯化,经SDS-PAGE分析可得单一的目的条带。NC膜皮下包埋法免疫效果良好,通过细胞融合、ELISA筛选获得3株能稳定分泌抗PRRSV N蛋白抗体的杂交瘤细胞株(H7、F7、C8),制备出高特异性的针对N蛋白的H7单抗。IFA与Western blotting结果显示制备的单抗可与病毒N蛋白产生特异性反应。亚型鉴定为IgG2b型,染色体分析证实杂交瘤细胞染色体数目正确。结果成功实现了N蛋白的原核表达,并获得高特异性的单克隆抗体,为PRRSV N蛋白抗原的检测奠定基础。     

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.     

The sustainability, feasibility and desirability of breeding livestock for disease resistance

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak.