Stable transfection of reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.     

水牛促卵泡素受体(FSHR)基因启动子克隆、生物信息学分析及转录活性检测

试验旨在对广西本地水牛FSHR基因5'侧翼序列进行克隆、生物信息学分析及其转录活性检测。根据GenBank已公布的黄牛FSHR 5'侧翼序列,本试验设计引物,以广西本地水牛血液基因组为模板扩增FSHR基因5'侧翼序列并进行生物信息学分析。结果显示本试验成功克隆了广西本地沼泽型水牛FSHR 5'侧翼序列及部分CDS区序列,共2979 bp,同源性比对分析结果表明其与河流型水牛、黄牛、绵羊、山羊、猪和人的同源性分别为100%、99%、93%、92%、91%和75%。对其5'侧翼2000 bp序列进行启动子预测及转录因子结合位点预测,结果显示在其翻译起始位点上游-147 bp附近存在TATA box,启动子区存在GATAs、FOXO1、FOXO3、Nobox、STAT1、STAT3、STAT4、STAT5A、STAT5B、STAT6和YY1等反式作用元件结合位点,其中GATAs家族基因在FSHR启动子区存在多个结合位点,且同一位点又存在多个GATAs家族基因结合的情况。水牛FSHR启动子能启动EGFP在HEK-293T细胞系中的表达,但表达非常微弱;也能启动EGFP在CHO细胞系中表达,且与CMV启动EGFP在CHO细胞系中的强度相似,结果表明水牛FSHR是个强启动子。总之,本研究成功克隆了沼泽型水牛FSHR基因启动子,分析了其启动子序列特征并成功验证其组织特异性的转录活性,为后期水牛繁殖性能分子机理阐明及基于卵巢特异性表达外源基因的转基因水牛奠定了基础。     

Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice

Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose significant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identification of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele.The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed.     

Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis

Major histocompatibility complex (MHC) class II antigen expression in hepatocytes and its correlation with mononuclear cell infiltration into the liver were studied using immunohistochemical techniques in 38 Dobermans with Doberman hepatitis (DH). Liver biopsy samples were obtained from 18 dogs at the subclinical stage. Autopsy samples were taken from 6 DH dogs euthanized for a reason other than DH, from 14 dogs euthanized because of advanced liver failure and from 6 control Dobermans. Upon examination of the control liver samples, no expression of MHC class II antigens was detected in hepatocytes. By contrast, in 15 of the 18 DH biopsies (83%) and in all 20 DH autopsy liver samples, hepatocytes expressed MHC class II molecules. MHC class II expression was either cytoplasmic or membranous and occurred in conjunction with lymphocyte infiltration. A correlation between the inflammatory reaction and the expression of MHC class II in hepatocytes suggests that the aberrant expression of MHC class II in hepatocytes is induced by cytokines. Hepatocytes presenting a putative MHC class II molecule-associated autoantigen could thus become the target of an immune attack mediated by CD4+ T cells. In addition, corticosteroid treatment was observed to significantly decrease MHC class II expression in DH hepatocytes. Inappropriate MHC class II expression in hepatocytes and mononuclear cell infiltration are suggesting an autoimmune nature for chronic hepatitis in Dobermans.     

The development of methods for immunophenotypic and lymphocyte function analyzes for assessment of Southern sea otter (Enhydra lutris nereis) health

The Southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act. The population began a pattern of slow decline in 1995. The decline was attributed to high adult mortality rates with infectious disease being the major cause of death. Multiple pathogens were implicated in these deaths including opportunistic pathogens such as Coccidiodes immitis and Toxoplasma sp. These findings suggested that the immunological health of mature animals in this population might be compromised. The primary goal of this study was to establish techniques for assessing phenotypic and functional baseline data for peripheral blood mononuclear cells (PBMC) in free-ranging sea otters. Standard total and differential white blood cell counts were augmented by emumeration of T and B lymphocyte subsets. Lymphocyte function was determined by both mitogen-induced proliferation and expression of IL-2 receptors. In addition to establishing normal ranges for adult animals, age-related changes were identified in B lymphocyte numbers and cell-surface density of major histocompatability complex class II (MHC II) proteins. The predominant lymphocyte subpopulation in Southern sea otters is the T lymphocyte. Substantial variation among individual animals was observed within the B lymphocyte population both in cell number and density of MHC II expression. Pups had greater numbers of T and B lymphocyte, as well as, greater MHC II expression on B lymphocytes than adults. Mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) was variable among individual animals with no significant difference in cell response between age class and gender. Concanavalin (ConA) was a more effective mitogen in stimulating proliferation and interleukin (IL)-2 receptor expression than pokeweed. This data can be used to augment routine hematology profiles and aid in the identification of animals with immunologic perturbations.     

Cloning and Sequence Analysis of MHC Ⅰ β2m Gene in Chicken

In order to further understand molecular characteristics and immune mechanisms of chicken major histocompatibility complex class Ⅰ(MHCⅠ) β2m gene,the 15 sequence of chicken MHC β2m gene from three local breeds was cloned by RT-PCR. We compared these sequences with seven MHCⅠβ2m genes of human,mouse and other animals in the GenBank database. The results showed that the amino acid homology among chicken MHCⅠβ2m ranged from 98.0% to 100%,the most sequences were identical. Chicken MHCⅠβ2m shared a 60.6% amino acid homology with duck,the highest degree of homology indicated a close genetic relationship,and the grasscarp had the lowest homology,33.3%. It could be further confirmed by phylogenetic analysis. Moreover,alignment of 10 β2m sequences of mature protein,two cysteines were located in sites 24 and 79,and there was the "YXCXVXH" Ig-motif character between the sites 77 and 83 of chicken β2m. The results showed that MHCⅠβ2m of chicken and other species had the same basic unit of immune function,and they had the unique structural features.     

Comparison of the accessory activity of murine peritoneal cavity macrophage derived dendritic cells and peritoneal cavity macrophages in a mixed lymphocyte reaction.

A detailed comparison of the accessory cell activities was carried out among murine peritoneal cavity macrophages (PEC-Mphi), peritonea] cavity macrophages stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4), the most popular cytokine combination widely used to generate dendritic cells (DC) and peritoneal cavity macrophage-derived DC (PEC-DC) using a two-way mixed lymphocyte reaction (MLR). All the cell types used efficiently induced statistically significant na?ve T cell proliferation at all culture time points and responder:stimulator ratios used. However, marked differences were noted in the magnitude of the proliferative responses. These variations may be attributed to the intensity of expression of MHC class II glycoproteins, as well as the actual numbers of MHC class II+ cells.     

鸡肝脏型脂肪酸结合蛋白基因启动子活性分析

PCR扩增鸡L-FABP基因5′侧翼区约2kb的DNA片段,进行克隆并测序,构建了鸡L-FABP基因报告基因系列缺失载体,瞬时转染进入人肝癌细胞系,利用双荧光素酶报告基因系统测定了荧光素酶活性。在线分析软件发现鸡L-FABP基因启动子区存在HNF-1、SREBP-1、AP-1、C/EBP、Oct-1、TATA、CCAAT、GATA-1等调控元件,没有发现CpG岛。报告基因结果表明鸡L-FABP基因启动子-2 076bp/-20bp区域具有最强的启动子活性,-522bp/-20bp区域启动子活性最弱;C/EBPα可以显著的抑制鸡L-FABP基因的表达,这些结果为深入研究鸡L-FABP的表达调控机制奠定了基础。     

Characterization of BF2 and beta2m in three Chinese chicken lines

Twenty-four BF2 genes and 10 beta(2)m genes from Chinese Sanhuang (SH), Wuji (WJ), and Zhenzhu (ZZ) chicken lines were cloned, and the amino acid replacement rates of the BF2 polypeptide binding domain were investigated. For this purpose, 13 BF2 genes from the SH-chicken line (BF2*01sh-BF2*13sh), six BF2 genes from the WJ-chicken line (BF2*01wj-BF2*06wj), and five BF2 genes from the ZZ-chicken line (BF2*01zz-BF2*05zz) were analyzed. The overall conservation of BF2 alleles could be observed within the sequences, and relative conservation was also displayed in the peptide-binding domain, CD8(+) interaction sites, and beta(2)m contact sites. Based on the amino acid similarity, BF2 from the three chicken lines could be divided into eleven gene groups, and five novel gene groups were observed. Although the amino acid similarity among the different alleles was 75.7-99.2%, within an allelic group the members shared >91% amino acid identity with each other. In addition, beta(2)m genes from the three Chinese chicken lines were also clustered into two gene groups: I and II. Between groups I and II, the amino acid identical ratio was much lower (81.9-84.0%). Group I is close to that of the reported chicken beta(2)m, whereas group II represents a new allelic group. The results suggest that five new BF2 groups and a new beta(2)m group exist in the three Chinese chicken lines.     

Gene organization and polymorphism of the swine major histocompatibility complex

The recent availability of the full‐length sequence of one haplotype of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), and significant progress in the studies on gene expression and polymorphisms led to major advances in deciphering its role in resistance to diseases in animals. The present status of the genomic organization and polymorphism of the SLA complex is presented in this Review. Additionally, a comparative analysis with mammalian MHC has also been provided. The sequenced SLA‐H01 haplotype harbors 152 loci including genuine SLA genes, non‐MHC genes and pseudogenes. Although the numbers of expressed SLA genes could vary across haplotypes, three SLA class Ia, three SLA class Ib, four SLA class IIa and four SLA class IIb genes are currently expressed. Except for the class I genes, which have no clear orthologs, the gene organization of the loci was highly conserved between humans and pigs. Moreover, the human leukocyte antigen (HLA) complex lies on a single chromosomal segment, whereas a centromere at the class II and III junction splits the SLA complex into two segments, without disturbing gene organization or impeding functionality. Over 400 SLA class I and II allele sequences available in databases have been recently clustered and assigned to a specific SLA locus according to a newly defined nomenclature system.     

Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins

Cell-mediated immune responses are important for protective immunity to Marek’s disease (MD), especially because MD herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC): B²¹B²¹) and RECC-CU91 (MHC: B¹⁹B¹⁹). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes obtained from MD-resistant N2a (MHC: B²¹B²¹) and MD-susceptible P2a (MHC: B¹⁹B¹⁹) chickens, indicating that the expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.     

Molecular cloning, polymorphism and tissue distribution of the MHC class IIB gene in the Chinese goose (Anser cygnoides)

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5'-RACE sequences (Ancy-MHC II5'-1, Ancy-MHC II5'-2, Ancy-MHC II5'-3), one 3'-RACE sequence (Ancy-MHC II-3') and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.     

miRNA-200a通过靶向ZEB1参与调节鸡胸肌细胞增殖分化

ZEB1在细胞增殖分化中发挥关键作用,然而关于ZEB1在鸡胸肌细胞增殖分化过程中的功能及其与miRNA互作的研究极少。为探索miRNA如何通过靶向ZEB1参与调节鸡胸肌细胞增殖分化,实验检测了ZEB1在55周龄和20周龄鸡胸肌组织中的表达,使用Target Scan及miRDB在线软件预测鸡ZEB1基因的靶向miRNA,构建ZEB1野生型、突变型双荧光报告载体,并在DF1细胞中验证了ZEB1的靶向miRNA,双荧光素酶报告实验结果说明miR-200a通过特异性结合ZEB1 3'非编码区种子序列直接靶向并抑制ZEB1基因的表达。结果表明:由于miR-200a在55周龄鸡胸肌表达下调,对ZEB1的抑制作用减弱,导致ZEB1在55周龄固始鸡胸肌组织表达较20周龄显著升高(P0.01)。本研究首次在鸡上证明miR-200a是ZEB1的靶向miRNA,且miR-200a可能通过靶向ZEB1参与调节鸡胸肌细胞的增殖分化,为深入理解miRNAs在家禽及其他鸟类中的分子调节机制提供了基础与依据。     

Major histocompatibility complex class I is downregulated in Marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon

The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor-ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Marek's disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect.     

Effects of Compound Chinese Herbal Medicinal Polysaccharide Extraction on NF-κB,TNF-α and IL-6 mRNA Expression in Different MHC B-Lβ Ⅱ Genotype of Chickens Lymphocyte

In order to explore the effect of compound Chinese herbal medicines polysaccharides(cCHMPS) on immunomodulatory in different MHC B-Lβ Ⅱ genotype chickens,200 White feather broiler were chosen and PCR-SSCP technique was applied to analyze the polymorphism of MHC B-Lβ Ⅱ gene. The peripheral blood were collected according to the different MHC B-Lβ Ⅱ genotype,and the lymphocytes were isolated from peripheral blood and the cCHMPS were added with a final concentration of 100,75,50 and 0 μg/mL for co-culturing 24 h.Then the expression of NF-κB,TNF-α,IL-6 mRNA in lymphocyte using Real-time PCR method were detected. The results showed that:Compared with the control group,different does of cCHMPS could significantly improve NF-κB,TNF-α,IL-6 mRNA expression levels in chicken with different MHC B-Lβ Ⅱ genotypes (P<0.05), and when the cCHMPS concentration was 50 μg/mL,the NF-κB,TNF-α,IL-6 mRNA expression levels in lymphocyte of AB and AA genotype chicken were significantly higher than that of other groups (P<0.05).The NF-κB,IL-6 mRNA expression levels of AC genotype chicken were significantly higher than the other groups (P<0.05). The TNF-α mRNA expression levels of AC genotype chicken were significantly higher than the other groups when cCHMPS was 100 μg/mL (P<0.05).There results indicated that the cCHMPS could stimulate NF-κB,TNF-α,IL-6 mRNA expression in different MHC B-Lβ Ⅱ genotype chickens,and the optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chicken.     

Effect of Compound Chinese Herbal Medicinal Polysaccharides on MyD88 Dependent Pathway Mediated by TLR4 in Lymphocytes of Chicken with Different MHC B-LβⅡ Genotypes

To investigate the effects of different doses of compound Chinese herbal medicinal polysaccharides (cCHMPS) on TLR4 and downstream MyD88 dependent signal transduction pathway components in chicken lymphocytes of different MHC B-LβⅡ genotypes,PCR-SSCP technique was applied to group layer according to different MHC B-LβⅡ genotypes.The peripheral blood lymphocytes of chicken with different MHC B-LβⅡ genotypes were collected,and added with 100,75,50 and 0 μg/mL cCHMPS (high,middle and low dose groups and control group),respectively,then co-culturing for 16,24,32 and 48 h.The expression of TLR4,MYD88 and TRAF-6 mRNA were detected using Real-time PCR method.The results showed that compared with control group,cCHMPS could significantly improve the expression levels of TLR4,MYD88 and TRAF-6 mRNA of different MHC B-Lβ Ⅱ genotypes chickens (P < 0.05);The expression levels of TLR4,MYD88 and TRAF-6 mRNA of AA genotype chicken lymphocyte in middle and low dose groups were higher than those of high dose group (except TLR4 gene cultured for 16 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BB genotype in high dose group were higher than those of other dose groups (except TLR4 gene cultured for 32 and 48 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BC genotype in low dose were higher than that of other dose groups (except TLR4 gene cultured for 16 h).There results indicated that cCHMPS played an important role in the body’s immune regulatory mechanism by binding to TLR4 in the surface of lymphocytes,activating the downstream MyD88-dependent signal transduction pathway,regulating cellular immunity,and cCHMPS optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chickens.