Cloning, characterization, and expression analysis of a thioredoxin from orange-spotted grouper (Epinephelus coioides)

Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.     

Identification and characterization of DSCAM isoforms isolated from orange-spotted grouper Epinephelus coioides

The Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin (Ig) superfamily member, was first identified from human and subsequently isolated from both vertebrates and invertebrates. Recent studies have shown that the DSCAM molecule serves diverse functions in neurodevelopment, such as axon guidance and neuronal migration. Most studies on DSCAM, however, have focused on mammals and arthropods, and our present knowledge of bony fish DSCAM is still limited. In this study, orange-spotted grouper Epinephelus coioides was used as an animal model to explore the possible functions of DSCAM. Two DSCAM isoforms were isolated, namely EcDSCAM A and EcDSCAM B, with lengths of 1648 and 2025 amino acids, respectively. The classical domain structure (i.e. 9Ig-4FNIII-1Ig-2FNIII-Transmembrane domain-Cytoplasmic tail) was also found in the coding regions of these two EcDSCAMs. Phylogenetic analysis showed that in the vertebrate DSCAM clade, the EcDSCAMs and various teleost DSCAMs were clustered into a subclade. Real-time PCR revealed that EcDSCAM B is the major EcDSCAM isoform, with the expression of EcDSCAM B being significantly higher than that of EcDSCAM A. During the first 14days after hatching (dph), increases in the expression of the two EcDSCAMs were observed at 2-4 and 8-11dph. EcDSCAM is expressed mainly in the intestine, nerve-related tissues, and stomach. Optic nerve transection analysis showed that EcDSCAM was up-regulated during optic nerve regeneration after optic nerve injury. We also investigated whether DSCAM expression was affected by viral nervous necrosis (VNN) disease or vibriosis. We found that when grouper were challenged with nervous necrosis virus (NNV), there were no meaningful changes in DSCAM expression, but challenge with Vibrio anguillarum led to a decrease in EcDSCAM levels in the brain. This decrease may be related to the pathogenesis of V. anguillarum.     

Identification and characterization of a MBP isoform specific to hypothalamus in orange-spotted grouper (Epinephelus coioides)

Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role in forming and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI. The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oligodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution.     

Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides

Orange-spotted grouper iridovirus (OSGIV) was the causative agent of serious systemic diseases with high mortality in the cultured orange-spotted grouper, Epinephelus coioides. Here we report the complete genome sequence of OSGIV. The OSGIV genome consists of 112,636 bp with a G+C content of 54%. 121 putative open reading frames (ORF) were identified with coding capacities for polypeptides varying from 40 to 1168 amino acids. The majority of OSGIV shared homologies to other iridovirus genes. Phylogenetic analysis of the major capsid protein, ATPase, cytosine DNA methyl transferase and DNA polymerase indicated that OSGIV was closely related to infectious spleen and kidney necrosis virus (ISKNV) and rock bream iridovirus (RBIV), but differed from lymphocytisvirus and ranavirus. The determination of the genome of OSGIV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the OSGIV and may provide useful information to develop diagnosis method and strategies to control outbreak of OSGIV.     

Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including β-propiolactone (BPL) inactivated virus at 4°C for 12h and formalin inactivated virus at 4°C for 12d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.     

Interleukin-1beta gene in orange-spotted grouper, Epinephelus coioides: molecular cloning, expression, biological activities and signal transduction

IL-1beta, a key mediator of inflammation, orchestrates a variety of immune responses by initiating gene expression. Herein, we have cloned and sequenced the IL-1beta in orange-spotted grouper (Epinephelus coioides), produced soluble mature recombinant IL-1beta in Escherichia coli, and characterized its biological properties and downstream signal transduction. The grouper IL-1beta cDNA was 1364bp in length, containing an open reading frame of 765bp. The predicted protein of 254 amino acids revealed the presence of the IL-1 family signature motif and the absence of a conventional ICE cut site. Phylogenetically, the grouper IL-1beta clustered closely with those of teleost belonging to Perciformes and apart from those of mammals. The grouper IL-1beta was constitutively expressed in almost all tissues examined, and was augmented in PBL after the addition of LPS or Poly I:C in vitro. The prokaryotically produced rIL-1beta significantly stimulated the proliferation of grouper head kidney cells, and activated gene expression of IL-1beta and COX-2. Moreover, the rIL-1beta-induced IL-1beta and COX-2 expression were reduced by p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125), respectively. Taken together, the present study indicated that grouper IL-1beta may have an important role in grouper immune system and activate similar downstream cascades as its mammalian counterparts.     

Molecular and immunohistochemical characterization of B-cell lymphoma-2-negative follicular lymphomas

Aberrant expression of the antiapoptotic protein BCL (B-cell lymphoma)-2 in neoplastic germinal centers is one of the diagnostic hallmarks of follicular lymphoma. If BCL-2 cannot be detected by immunohistochemistry, the distinction between florid follicular hyperplasia and follicular lymphoma might become a diagnostic challenge. Most of those cases also lack the typical t(14;18), and the underlying pathophysiologic conditions of follicular lymphoma that lack BCL-2 protein expression are largely unknown. Here, we collected 18 BCL-2-negative follicular lymphoma cases from 5 different institutions. After restaining, 9 cases proved to be truly BCL-2 negative (6 follicular lymphoma grade 2, 2 follicular lymphoma grade 3a, and 1 follicular lymphoma grade 3b). In 4 additional cases, BCL-2 was very faint (all grade 2). Of the 9 BCL-2-negative follicular lymphoma cases, 2 were negative for CD10 (22%); all showed expression of BCL-6. Apoptotic level as determined by caspase 3 was the lowest in the BCL-2-positive follicular lymphoma group (15 ± 8 mm(2)), the highest in the normal/reactive group (n = 7, 60 ± 12 mm(2)) and very similar in the BCL-2 low follicular lymphoma and BCL-2-negative follicular lymphoma groups (25 ± 13 and 33 ± 19 mm(2), respectively), assuming an intermediate position between reactive follicles and BCL-2-positive neoplastic follicles (P < .001 [Kruskal-Wallis]). Also noted was a difference in proliferation fractions between the BCL-2-positive follicular lymphoma (27% ± 15%), the BCL-2 low follicular lymphoma (30% ± 20%) and the BCL-2-negative follicular lymphoma groups (30% ± 22%). Regarding the network of follicular dendritic cells, 8 (89%) of 9 cases from the BCL-2-negative subgroup showed disrupted, weakly developed networks, whereas all of the follicular lymphoma BCL-2 low-expression cases showed a well-defined and strongly developed follicular dendritic cell network. Among BCL-2-negative follicular lymphoma, BCL-2 and BCL-6 breaks were found in 1 case each, whereas in the follicular lymphoma BCL-2 low group, only 1 case with a BCL-6 break was recorded. No statistically significant result was achieved upon assessment of BCL-2α or BCL-2β RNA or the ratio of α/β isolated by real-time-polymerase chain reaction. Taken together, BCL-2-negative follicular lymphoma did not show a BCL-2 break on the genetic level and showed both increased apoptotic and proliferation rates compared with BCL-2-positive follicular lymphoma. In our series, BCL-6 breaks were infrequent in BCL-2-negative follicular lymphoma.     

Identification and functional characterization of an interferon regulatory factor 7-like (IRF7-like) gene from orange-spotted grouper, Epinephelus coioides

Interferon regulatory factor (IRF) 7 plays a crucial role in modulating cellular responses to viral infection and cytokines, including interferons (IFNs). In the present study, a novel IRF7 gene (designated as EcIRF7) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcIRF7 cDNA is composed of 2089 bp and encodes a polypeptide of 433 amino acids with 81% identity to IRF7 of Siniperca chuatsi, and the genomic DNA of EcIRF7 consists of 9 exons and 8 introns, with a length of approximately 5629 bp. EcIRF7 contains three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain, all of which are highly conserved across species. Recombinant EcIRF7 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcIRF7 serum preparation. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcIRF7, with a relative strong expression in spleen, kidney, skin and intestine. The expression of EcIRF7 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus and Singapore grouper iridovirus (SGIV). EcIRF7 showed similar intracellular localization pattern to those of mammalian and chicken, and translocated into nucleus after SGIV infection. Further more, EcIRF7 was proved to be capable of activating zebrafish type I IFN promoter and inhibiting the replication of SGIV in grouper spleen (GS) cells. These results suggest that EcIRF7 is potentially involved in grouper immune responses to invasion of viral and bacterial pathogens.     

人Bcl-2蛋白的基因克隆、表达及初步活性分析

目的：以特定形式对高度疏水性的人Bcl-2(hBcl-2)蛋白进行可溶性表达，获得非疏水性hBcl-2蛋白并具备生物学结合活性，为进一步研究hBcl-2三维结构并在此基础上研发特异性小分子药物提供充足的蛋白样品。方法：用PCR方法对hBcl-2基因进行克隆重组，插入原核表达载体pET28a( )中。用Western Blot和MALDI-TOF生物质谱鉴定，采用荧光极化方法进行活性测定。结果：重组hBcl-2在E．Coli中实现了高效、可溶性的融合表达，重组蛋白经纯化至电泳纯，具备了与Bcl-2家族Bak蛋白BH3功能域特异结合的能力，表明原核表达的重组hBcl-2具有较好的结合活性。结论：成功地对重组hBcl-2蛋白进行了可溶性表达和活性测定。     

Molecular cloning,expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442 bp and contained an open reading frame of 780 bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.     

Molecular cloning,structural modeling,and expression analysis of MyD88 and IRAK4 of golden pompano (Trachinotus ovatus)

MyD88 and IRAK4 are important components of TLR signaling pathways. However, information about MyD88 and IRAK4 is vacant in golden pompano (Trachinotus ovatus), a marine teleost with great commercial value. Thus, in this study the full lengths of trMyD88 and trIRAK4 were cloned from golden pompano using RT-PCR and RACE-PCR methods. trMyD88 was 1213 bp in length, encoding a putative protein of 288 amino acids (aa), consisting of a 99 aa of death domain at its N-terminal and a 137 aa of the TIR domain at its C-terminal. trIRAK4 was 1606 bp in length, encoding a putative protein of 469 aa, including an N-terminal death domain and a central kinase domain, connected by a ProST domain. Other domains or aa residues needed for their functions were also identified in trMyD88 and trIRAK4. Physicochemical features and 3-D structures of trMyD88 and trIRAK4 were also analyzed. Quantitative real-time PCR revealed that the 2 genes were ubiquitously expressed in tissues from healthy pompano, especially highly in the spleen and head kidney, indicating their roles in the immune response. Further, trMyD88 and trIRAK4 were up-regulated at 12 h after the Vibrio alginilyticus and polyI:C challenge and continued to 48 h post challenge. Our results demonstrated that MyD88 and IRAK4 played important roles in the golden pompano innate immune system, providing the basis for further study of the signaling pathways that these 2 genes are involved in.     

Survivin、Bcl-2在结肠癌组织中的表达及意义

目的 探讨Survivin、Bcl-2在结肠癌组织中的表达及其与结肠癌临床病理学特征及预后的关系。方法 利用免疫组织化学染色方法检测55例结肠癌组织、33例癌旁结肠组织及15例正常结肠癌组织中Survivin、Bcl-2蛋白的表达情况,结合临床病理特征及预后对其进行分析。结果 Survivin、Bcl-2在结肠癌组织中的表达率显著高于癌旁组织和正常结肠组织中的表达率（P < 0.05）。有淋巴转移的癌组织中Survivin、Bcl-2表达率显著高于无淋巴转移的癌组织中的表达率（P < 0.05）。Survivin、Bcl-2的表达呈高度一致性（P < 0.05）。Survivin阳性组和Bcl-2阳性组患者的5年、10年累积生存率显著低于阴性组（P < 0.05）。结论 Survivin、Bcl-2与结肠癌的生物学行为密切相关,并可作为结肠癌患者预后的预测因素。     

LIGHT基因的克隆及其可溶性表达

目的　克隆LIGHT基因 ,构建含有人LIGHT基因的表达载体 ,诱导其在大肠杆菌中可溶性表达 ,并对表达的LIGHT蛋白的生物学活性进行检测。方法　从人的外周血单个核细胞中克隆LIGHT全长cDNA及其胞外区片段 ,并将其胞外区片段亚克隆至原核表达载体pET 11a中 ,筛选阳性重组质粒pET LIGHT ,以IPTG诱导其可溶性表达 ,并以SDS PAGE和Westernblot检测进行分析。表达的蛋白初步纯化后 ,进行生物学活性分析。结果　RT PCR扩增出了LIGHT全长 72 3bp的cDNA。SDS PAGE和Westernblot分析证实重组pET LIGHT质粒可表达出相对分子质量 (Mr)为 19× 10 3的蛋白。可溶性LIGHT重组蛋白可共刺激T细胞的增殖及诱导IFN γ的产生。结论　本实验成功地将LIGHT胞外区片段在大肠杆菌中进行表达 ,表达的蛋白具有生物学功能 ,这为进一步的LIGHT基因的功能研究打下了基础     

hTRT催化功能区基因克隆及其在肿瘤中的表达

目的 研究端粒酶蛋白ｈＴＲＴ基因在肿瘤中的表达及其意义。方法 用ＲＴ－ＰＣＲ法检查Ｈｅｌａ细胞与ＰＧ细胞的ｈＴＲＴ表达水平,将Ｈｅｌａ细胞的ｈＴＲＴ催化功能克隆,并进行测序比较,应用原位杂交技术对肿瘤组织中的ｈＴＲＴ和ｈＴＲ（端粒酶ＲＮＡ组分）基因进行检测。结果 Ｈｅｌａ细胞与ＰＧ细胞均有ｈＴＲＴ表达,Ｈｅｌａ细胞ｈＴＲＴ催化功能区ｃＤＮＡ序列与文献报道一致,原位杂交结果显示ｈＴＲＴ与ｈＴＲ的协同