人干扰素调节因子3基因启动子的初步鉴定

子活性分析表明,IRF3启动子区域定位于主要转录起始位点区域前111-167 bp的区域内.采用转录因子结合位点预测分析软件分析表明,该区域内存在E2F转录因子结合位点.结论 -167～-111 bp区存在IRF3启动子的核心调控元件,转录因子E2F可能参与IRF3的转录调控.     

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.     

小鼠与人重排活化基因2启动子的比较

目的 通过比较小鼠与人重排活化基因2(RAG2)启动子，试图寻找与小鼠RAG2启动子特异性结合的转录因子。方法 采用表达luciferase的报告基因载体检测启动子的活性。采用：EMSA(electrophoresis mobility shift assay)检测与启动子结合的转录因子。结果小鼠：RAG2启动子-60／-41区域存在富G的GA盒子，而人RAG2启动子在相应位置却是富A区。突变实验结果显示，GA盒子是小鼠RAG2启动子完整活性所必须的。EMSA结果显示，Spl／Sp3结合在小鼠RAG2启动子-60／-41区域，并且Spl能够协同Pax-5、c-Myb活化小鼠RAG2启动子。结论 尽管小鼠与人RAG2启动子同源性很高，但它们结合的转录因子和功能有所不同。     

Trappin‐2/Elafin: a novel innate anti‐human immunodeficiency virus‐1 molecule of the human female reproductive tract

Trappin‐2/Elafin is a serine protease inhibitor that plays a major role as an anti‐inflammatory mediator at mucosal surfaces. In addition, Trappin‐2/Elafin has antibacterial activity against Gram‐positive and Gram‐negative bacterial and fungal pathogens. In this study we examined the production of Trappin‐2/Elafin by epithelial cells from the human upper and lower female reproductive tract as well as its activity as an anti‐human immunodeficiency virus (HIV)‐1 molecule. We found that primary uterine, Fallopian tube, cervical and ectocervical epithelial cells produce Trappin‐2/Elafin constitutively and that production of Trappin‐2/Elafin is enhanced following stimulation with Poly(I:C), especially by the uterine cells. Given the presence of Trappin‐2/Elafin in the reproductive tract, we tested the ability of recombinant Trappin‐2/Elafin to inhibit HIV‐1, an important sexually transmitted pathogen. We found that recombinant Trappin‐2/Elafin was able to inhibit both T‐cell‐tropic X4/IIIB and macrophage‐tropic R5/BaL HIV‐1 in a dose‐dependent manner. The inhibitory activity was observed when virus was incubated with Trappin‐2/Elafin but not when Trappin‐2/Elafin was added to cells either before infection or after infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV‐1 and Trappin‐2/Elafin. Additionally, we measured the levels of secreted Trappin‐2/Elafin in cervico‐vaginal lavages (CVL) from both HIV‐positive and HIV‐negative women and found that average levels of secreted Trappin‐2/Elafin were higher in the CVL from HIV‐negative women, although the values did not reach statistical significance. We also found that women at the secretory phase of the menstrual cycle produced more Trappin‐2/Elafin in CVL relative to women at the proliferative phase of the menstrual cycle. Our data suggest that Trappin‐2/Elafin might be an important endogenous microbicide of the female reproductive tract that is protective against HIV‐1.     

Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon gene and characterisation of its expression in response to nodavirus infection

We cloned and sequenced 2C I-IFN, a two-cysteine containing type I interferon (I-IFN) gene, in orange-spotted grouper (Epinephelus coioides). The cDNA has 769 base pairs, the protein has 172 amino acids, and the predicted signal peptide has 18 amino acids with two cysteines. This gene is similar to I-FNs from sea bass and other teleosts. 2C I-IFN has 5 exons and 4 introns, also similar to other teleost I-IFNs. Immunohistochemical (IHC) analysis indicated that expression is predominantly membrane-localized in healthy grouper, but has a zonal distribution in nodavirus-infected grouper. Grouper infected with nodavirus had elevated levels of 2C I-IFN at 72 h and Mx at days 6–7. Recombinant 2C I-IFN activated grouper Mx, leading to upregulated antiviral activity. The grouper Mx promoter was highly induced after treatment with recombinant 2C I-IFN. The present results suggest that expression of grouper 2C I-IFN may participate in the immunologic barrier function against nodavirus.     

Expression and clinical significance of B cell translocation gene 2 in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is widely known as a highly fatal cancer, and thus it is important to identify tumor-specific and radiosensitivity-specific markers in ESCC. B cell translocation gene 2 (BTG2) has been considered a novel tumor suppressor gene or radiotherapy sensitivity-associated gene. However, the relationship between BTG2 and ESCC development and radiotherapy sensitivity is uncertain. The present study aims to explore the expression and clinical significance of B cell translocation gene 2 (BTG2) in ESCC by analyzing the RNAseq data from the TCGA and immunohistochemical staining of ESCC samples. We found that the level of BTG2 mRNA was significantly decreased in ESCC patients, and further decreased significantly in radiotherapy resistant patients compared to sensitive patients. The positive expression rate of BTG2 protein was 56.0% (103/184) in 184 ESCC tissue samples and 84.0% (42/50) in normal esophageal mucosal samples, respectively. The positive ratios of BTG2 expression in radiotherapy-sensitive group and radiotherapy resistant group were 57.9% (22/38) and 23.5% (4/17), respectively. Furthermore, the analysis indicates that the expression level of BTG2 significantly correlated with lymph node metastasis and clinical staging in ESCC patients. A multivariate analysis with Cox regression model showed that BTG2 level was an independent risk factor affecting the prognosis of ESCC patients. Above all, the downregulation of BTG2 may be used as a molecular marker to identify and predict ESCC progression and radiosensitivity.     

Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether Hand2 affects neurogenesis and/or expression of vasoactive intestinal polypeptide and choline acetyltransferase in developing enteric neurons. Gain-of-function of Hand2 in HNK-1(+) immmunoselected precursor cells resulted in increased neurogenesis. The number of neurons expressing vasoactive intestinal polypeptide increased in response to Hand2 overexpression although choline acetyltransferase was not affected. Targeted deletion of Hand2 in neural crest cells resulted in loss of all neurons expressing vasoactive intestinal polypeptide along the length of the gastrointestinal tract, patterning defects in the myenteric plexus of the stomach, and altered number and morphology of neurons expressing TH. Our data demonstrate that expression of Hand2 is sufficient and necessary for neurogenesis and expression of a subset of cell type-specific markers in the developing enteric nervous system.     

Expression of a novel complement C3 gene in the razor clam Sinonovacula constricta and its role in innate immune response and hemolysis

Complement component 3 (C3) is a core component of the complement system, and directly participates in immune regulation and immune defense. Isoforms of C3 have been reported in several species of vertebrate, but invertebrates, and more specifically clams, have been less well studied. An isoform of C3, named ScC3-2, was identified in Sinonovacula constricta (Chinese razor clam). ScC3-2 included eight conserved regions, a thioester bond and two predicted junction sites (α-β and α-γ). The gene was expressed in the liver, gill, foot, hemolymph, mantle, gonad and siphon tissues. The gene was significantly upregulated in umbo larvae, suggesting that initial larval immunity may develop in umbo larvae. Moreover, the ScC3-2 mRNA expression patterns after challenge with Vibrio parahemolyticus and Micrococcus lysodeikticus exhibited an obvious upregulation at 8 h in the hemolymph and at 4 h in the liver, respectively. Furthermore, ScC3-2 showed effective membrane rupture of heterologous rabbit erythrocytes. The ScC3-2 protein was located on the surface of the cells during the process of hemolysis. After a comparative analysis, we suggest that the major structure and function of ScC3 and ScC3-2 are analogous. Our findings suggest that ScC3-2 plays an important immune function, and an intricate complement response may exist in S. constricta.     

Developmental regulation of type 1 and type 3 interferon production and risk for infant infections and asthma development

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一株新的鼠抗人2IgB7-H3功能性单克隆抗体的研制及其生物学特性的鉴定

研制功能性鼠抗人2IgB7-H3单克隆抗体。以人2IgB7-H3基因转染细胞L929/2IgB7-H3为免疫原,常规免疫6~8周龄的雌性BALB/c小鼠;利用B淋巴杂交瘤技术,将免疫小鼠脾脏细胞和骨髓瘤细胞株SP2/0融合,L929/2IgB7-H3细胞为抗原筛选阳性细胞;经间接免疫荧光标记法对杂交瘤细胞进行反复筛选和多次克隆化培养;采用快速定性试纸法鉴定单抗Ig亚类;利用竞争抑制性实验分析该单抗识别的抗原表位;采用MTT法分析该单抗在体外阻断DC对T细胞的促增殖效应。结果:获得了1株持续稳定分泌鼠抗人2IgB7-H3单克隆抗体的杂交瘤细胞株(命名为7D7),该单抗可特异识别L929/2IgB7-H3分子和介导有效的共刺激信号。该单抗的成功获得和其生物学特性的初步鉴定,为进一步研究B7-H3两个异构体在DC细胞及肿瘤细胞上的作用机制奠定了物质基础。