Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.

Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.

Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stornatits Virus expressing green fluorescence protein (rVSV-GFP) system in the equine fetal kidney-78 cell line (EFK-78). The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with EIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titer of antiviral activity was 1 x 10(3) AU/ml. These results demonstrated that the EIFN-gamma expressed in this study had good biological activity. Pure forms and sufficient quantities of biologically active IFN-gamma could facilitate the study of its activities in modulating immune responses both in vivo and in vitro. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-γ gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of BALB/c mice with rEMIL-18, nine monoclonal antibodies (mAbs) against equine interleukin-18 (EIL-18) were obtained and characterized. These mAbs recognized different epitopes on equine mature interleukin-18 (EMIL-18) protein based on their reactivity with two peptides containing different amino acid sequences and one of these mAbs has neutralization activity against EIL-18 in an IFN-γ-induction assay.

OBJECTIVE To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice. METHODS Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression. RESULTS The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P<0.05), but lower level of antibody (P<0.05). CONCLUSION Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

In order to investigate the immune responses to the nucleoprotein(NP) of Avian influenza virus,NP gene cut from re-combinant plasmid pVAX-NP was subcloned into a eukaryotic expression vector pCAGGS with an avian-derived promoter.The re-combinant plasmid pCAGGS-NP was obtained after PCR identification and sequencing.Then the positive pCAGGS-NP was subject-ed to transfect eukaryocyte and to immunize SPF chickens.Indirect florescent assay(IFA) was engaged to detect the transient expres-sion of NP,and ELISA was used to detect the serum antibody of the immunized chickens which was resistant to NP protein.The re-sults showed that NP was successfully expressed both in vitro and in vivo and had good immunoreactivity.

OBJECTIVE:To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.METHODS:The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.RESULTS:PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).CONCLUSION:The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.

For the construction of BF2/peptide tetramer, large amount of recombinant chicken major histocompatibility complex (MHC) Ⅰ heavy chain (BF2-BSP) and light chain Chβ2m were expressed and purified. And a peptide originated from avian Infec- tious bronchitis virus (IBV) nucleoprotein (N71~78) was synthesized. Afterwards, BF2-BSP and Chβ2m were refolded with IBV N71~78 in refolding buffer to generate the monomer of BF2/peptide complex. Then the folded product was treated with enzymatic biotinylation by BirA enzyme and followed by a gel filtration column to purify the BF2/peptide fraction. Finally, the BF2/peptide tetramer was formed by mixing purified, biotinylated monomer with phycoerythrin(PE)-labeled streptavidin at a molar ratio of 5∶1. In order to test if the prepared BF2/peptide tetramer could be employed to detect the efficiency of specific T cell, the peripheral blood mononuclear cells (PBMCs) were isolated from the SPF chickens infected with IBV H52 at 10 days post infection and subjected to flow cytometry stain- ing. The cytotoxitic T lymphocytes (CTLs) response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% of CTLs frequency specific to IBV N was detected. All these results indicated that the prepared BF2/peptide tetramer can be used to evaluate the specific T cell immune responses of IBV.

Objective: To label equine IFN-γ monoclonal antibody (McAb) with fluorescein isothiocyanate (FITC) and provide an experimental material for intracellular cytokine staining (ICS) assay of IFN-γ in equine lymphocytes. Methods: Purified equine IFN-γ McAb SB10 was labeled with FITC, then diluted serially and used for ICS assay of IFN-γ in equine PBMCs by flow cytometry. The result was compared with that of flow cytometry by using commercial FITC-labeled bovine IFN-γ McAb CC302. Results: When 106 PBMCs were stained with 1 μl of FITC-CC302, the frequency of PBMCs secreting IFN-γ was 9. 35%. However, when 106 PBMCs were stained with 0. 25 μl of FITC-SB10, the frequency was 9. 90%. Conclusion: The prepared FITC-labeled equine IFN-γ McAb laid a material foundation of developing the methods for immunological study on equine cells and provided a technical platform for monitoring the immune state and investigating the immunological mechanism.

The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N1 virus A/VN/1203/2004 (clade 1) was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca) that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05) (clade 2.3), and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca). AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2). These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.

目的:建立检测马白细胞介素-18(equine interleu-kin-18,EIL-18)的双抗体夹心ELISA,为马传染病的免疫学研究提供新方法。方法:将识别不同表位的EIL-18的2株单克隆抗体(mAb)B1G1和S6A3纯化后,利用生物素标记试剂盒对B1G1株mAb进行标记,以重组马白细胞介素-18(rEIL-18)为捕获抗原进行ELISA检测来建立EIL-18双抗体夹心ELISA。结果:经过方阵滴定试验确定捕获抗体的最佳浓度为4mg/L,检测抗体的工作效价为1∶2000。建立的方法可检测马血清中的EIL-18,与猪、牛和羊血清中的IL-18不发生交叉性反应,此方法检测敏感度达到15ng/L,特异性良好。结论:成功建立了EIL-18的双抗体夹心ELISA,为马细胞免疫学研究提供了方法,也为下一步EIL-18ELISA试剂盒的商品化开发奠定了坚实基础。

To develop a multiplex RT-PCR method for simultaneous detection of RVFV,HEV,RV,VSV and FMDV,a set of specific primers were designed based on the published sequences.PCR products of 499 bp,137 bp,274 bp,224 bp and 389 bp were obtained for the respective viruses.The specificity of this method was evaluated by testing representative viruses in the same family and related zoonosis viruses and no PCR products were detected for NDV,AKAV,BEFV and JBEV.This method has a sensitivity of detecting as low as 2.91 ×104 (RVFV),3.14 ×103 (HEV),1.25 ×103 (RV),6.65 ×103 (VSV) and 2.38 ×104 (FMDV) copies of template RNA.Test on 11 RV samples and 7 HEV samples all produced positive results.

To examine the bioactivity of recombinant equine interleukin-18 (rEIL-18) obtained from Escherichia coli, rEIL-18 was evaluated to see if it showed a synergistic effect with recombinant human interleukin-12 (rhIL-12) for the induction of equine interfer-on-γ (EIFN-γ ) gene expression from equine peripheral blood mononuclear cells (PBMCs) by Real-time RT-PCR. REIL-18 could in- duce EIFN-γ mRNA expression from equine PBMCs about 20-fold higher than that of negative control in the presence of rhIL-12. And the effect to induce EIFN-γ gene expression was dose-dependent with rEIL-18 in a fixed concentration of rhIL-12. Nine an-ti-rEIL-18 monoclonal antibodies (mAbs) were also evaluated if they could neutralize the bioactivity of rEIL-18. And one of the 9 mAbs could neutralize rEIL-18 bioactivity and its effect was dose-dependent.

To obtain a recombinant glycoprotein G1 of Akabane virus(AKAV)for diagnostic purpose,the gene fragment coding high antigenic domain of AKAV G1 protein was amplified from the AKAV genome by RT-PCR after analyzing glycoprotein G1 with bio-software.The amplified product was cloned into pMD1S-T vector and sequenced.Then the gene fragment was subcloned into pET-28a(+)vector directionally.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).SDS-PAGE and Western blotting analysis indicated that the fusion protein was insoluble and approximately 44 kD in molecular weight and had immunological activity.After purified by affinity chromatography,the concentration of purified protein was 2 mg/mL and the purity was 92.6%.An indirect enzyme linked immunosorbent assay(ELISA)was developed using the purified recombinant protein.The optimal reaction conditions of ELISA were determined:the coating concentration of the purified recombinant protein was 20μg/mL; the dilution fold of the serum samples was 1:200.There was no cross reaction with positive sera of other six infectious diseases. Bovine serum samples from Yunnan(77)and Inner Mongolia(70)had been detected by serum neutralizing(SN)test before detected by the ELISA.In accordance with SN test,the positive threshold of the ELISA was determined as 0.493 and 0.488 in two districts respectively;the speciality of the ELISA was 73% and 86.9% respectively.And the agreement ratio between the two methods was 80%(52/65)and 85.9%(55/64)respectively.

The integrated application of multi-source and multi-temporal remote sensing data is the trend of remote sensing application research, and it is also the practical need to solve the inversion problem of remote sensing. In this paper, a method is developed to retrieve canopy biophysical variables using multi-temporal remote sensing data. The inherent change rules of biophysical variables are introduced into the retrieval methods by coupling the radiative transfer model with land process model to simulate time series surface reflectances. A cost function is constructed to compare the reflectances simulated by the coupled model with time series reflectances measured by sensors and the canopy biophysical variables with the available prior information. And an optimization method is used to minimize the cost function by adjusting the values of input canopy biophysical variables such as the temporal behaviour of the reflectances simulated reaches the best agreement with the multi-temporal reflectances measured. Retrieval of leaf area index from MODIS surface reflectance data (MOD09) at the Bondville site was performed to validate this method. The experimental results shows that the use of multi-temporal remote sensing data can significantly improve estimation of canopy biophysical variables. * Corresponding author.

在病毒感染机体时,MHC-I类分子限制的抗原特异性CTL反应是清除病毒、控制病毒复制和散播的主要机制[1].

Equine interferon-gamma (eIFN-gamma) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-gamma. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1 x 10(3) AU/mL and 1 x 10(5) AU/mL of eIFN-gamma expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-gamma were highly efficient and specific, as it was blocked by mAbs against eIFN-gamma.

采用RT-PCR技术从猪外周血单核细胞(PBMC)中获得猪白细胞抗原基因座P1、P14等位基因的胞外区,并在其3′端拼接上依赖BirA酶的可生物素化序列(BSP),构建了pET-P1-BSP和pET-P14-BSP高效表达载体并进行诱导表达和纯化,用SDS-PAGE和Western blotting法对表达产物进行鉴定.结果显示,成功构建了融合表达载体pET-P1-BSP和pET-P14-BSP,表达产物以包涵体的形式存在,获得高纯度的P1-BSP和P14-BSP蛋白,为进一步利用亲和素在体外构建SLA-I类分子肽四聚体奠定了基础.

BALB/c mice(Mus musculus) were immunized intraperitoneally with purified recombinant mEIFN-γ protein. Murine myeloma cells were fused with splenocytes of the immunized mice. An indirect ELISA with recombinant mEIFN-γ derived from baculovirus expression system as antigen was used to screen antibody-producing hybridomas. Twelve hybridomas cells could produce McAbs steadily after 3 cycles of subcloning. Additionally, all McAbs showed positive reaction to mEIFN-γ in indirect fluorescence assay (IFA) tests. Four McAbs were IgG1, two McAbs were IgG2a, three McAbs were IgG2b and three McAbs were IgM isotype, respectively. The light chains of all McAbs were chain. Furthermore, seven McAbs showed positive reaction to GST-mEIFN-γ(1~84) protein, and the others showed positive reaction to GST-mEIFN-γ(80~146) protein in specific ELISA tests. The results indicated that 12 McAbs could recognize two or more mEIFN-γ epitope. Therefore, these McAbs can be useful as a basis of detection methods for equine IFN-γ, monitoring immune state and investigating immunologic mechanism.