Identification of differentially expressed genes in Con A-activated carp (Cyprinus carpio L.) leucocytes

A cDNA library was constructed from the message RNA (mRNA) obtained from Con A-induced head kidney (HK) leucocytes of carp (Cyprinus carpio L.). Differential screening of the cDNA was carried out by hybridization against the total cDNA probes from normal, Con A-uninduced HK leucocytes or Con A-induced HK leucocytes of carp. The differential expression patterns of certain cDNA clones were confirmed by Southern-blot and Northern-blot analysis. Single-pass of the sequencing analysis and homology search in Genbank (EMBL) revealed those differentially expressed cDNA clones encode for cytochrome c oxidase sub-unit II and III (COII and COIII), elongation factor-1 beta (EF-1 beta), bleomycin hydrolase (BH), heat shock cognate protein 70 (HSC70) and 16S ribosomal RNA (16S rRNA).     

Characterisation of a monoclonal antibody to carp IL-1beta and the development of a sensitive capture ELISA

A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1beta was developed with a detection limit of 10 ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1beta in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1beta is known to stimulate proliferation of T & B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.     

Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1 beta, high affinity immunoglobulin E Fc receptor gamma subunit and serum amyloid A

Suppression subtractive hybridisation (SSH) is a powerful means to identify genes of cytokines and other genes that express small amount of mRNA. In this study, cDNA of normal fish (carp) head kidney cells (HKC) was subtracted from pooled cDNA of HKC and peritoneal cell (PC) obtained from fish which had been injected with sodium alginate (SA) and scleroglucan (SG) 3-48 h earlier. This subtraction produced 248 clones of cDNA fragments. After sequencing some of the fragments of interest were used as probes, and yielded full-length cDNAs homologous to mammalian interleukin-1 beta (IL-1 beta), the gamma subunit of high affinity Fc receptor for IgE (Fc epsilon RI gamma) and serum amyloid A (SAA); these were cloned and sequenced. Carp IL-1 beta shows 21.8-24.7% amino acid identities to mammalian mature IL-1 beta, and lacks a signal sequence, which is consistent with mammalian IL-1 beta. Carp Fc epsilon RI gamma, which was the first cloned non-mammalian Fc receptor subunit, shows 39.3-40.4% amino acid identities to mammalian Fc epsilon RI gamma, and contains the immunoreceptor tyrosin-based activation motif characteristic of the signal transduction subunit of antigen- and Fc-receptors. Carp SAA is most similar to mammalian acute phase responsive type SAA with 53.0-55.3% amino acid identities. Both SA-elicited and SG-elicited PC expressed higher amounts of IL-1 beta and SAA mRNA compared to saline-injected fish HKC and PC, indicating that these proteins are associated with inflammatory responses, similar to mammalian homologues. Fc epsilon RI gamma was constitutively expressed in leucocytes and not immunopotentiator-responsive, but this indicates that Fc receptor including Fc epsilon RI gamma subunit is likely functional in the carp immune system.     

草鱼诱导型一氧化氮合酶cDNA和启动子的克隆及分析

应用PCR和RACE技术,以细菌脂多糖(LPS)刺激的草鱼头肾白细胞cDNA为模板,获得草鱼iNOS (Inducible nitric oxide synthase)全长cDNA序列.该序列共4286 bp,编码含1080个氨基酸残基的蛋白.氨基酸序列比对分析发现草鱼iNOS与金鱼iNOS a和b、斑马鱼iNOS 2b以及鲤鱼iNOS高度保守,序列一致性均大于80%;它们的血红素、四氢生物蝶呤、钙调蛋白、FMN、FAD和NADH等辅因子结合区域也十分保守.用NJ法对新获得的序列和其它脊椎动物NOS的编码序列进行进化分析证实它属于诱导型NOS.在此基础上,运用基因步移技术分离草鱼iNOS基因的启动子序列,共有1978 bp.生物信息学分析发现该序列含有包括GR、AP1、C/EBP、ER、YY1、IRF1和IL-6 REBP在内的多个转录因子的潜在结合位点.     

Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor beta 4 from chicken embryo chondrocytes

Using a human transforming growth factor beta 1 (TGF beta) cDNA probe, we have detected an RNA species migrating at about 1.7 kilobases in cultured primary chicken embryo chondrocytes that is distinct from chicken TGF beta 1. The cloning and sequencing of cDNAs corresponding to this chondrocyte RNA demonstrate that it represents a new member of the TGF beta family, which we have named TGF beta 4. Unlike previously described TGF beta which are 390 to 414 amino acids long, the predicted precursor protein of TGF beta 4 is only 304 amino acids and does not appear to contain a signal peptide. Also unique to this new TGF beta is an insertion of two amino acids near the N-terminus of the processed peptide which would result in a 114 amino acid mature protein after cleavage from the precursor at a tetrabasic arg-arg-arg-arg site. The nine cysteine residues characteristic of all TGF beta are conserved. TGF beta 4 shows 82%, 64%, and 71% identity with the amino acid sequences of processed TGF beta 1, 2, and 3, respectively.     

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-beta messenger ribonucleic acid from chick embryo chondrocytes

Transforming growth factor beta 1 (TGF beta 1) has been purified and the mRNA cloned from a number of mammalian species including human, murine, bovine, porcine, and simian. Using a human TGF beta 1 cDNA probe, we have detected two distinct TGF beta RNAs in cultured primary chick embryo chondrocytes. One of these RNAs, migrating at about 1.7 kilobases, shows similarity to mammalian TGF beta 1. The second RNA, migrating at about 3 kilobases, is a novel TGF beta mRNA which we have named TGF beta 3. Clones corresponding to each of these RNAs were isolated from a cultured primary chick embryo chondrocyte cDNA library. Two cDNA clones for TGF beta 3, pTGFB-ChX17 and pTGFB-ChX25, contained a 39 nucleotide-long 5'-untranslated region, a 1236 nucleotide-long coding region, and a 911 nucleotide-long 3'-untranslated region. The predicted protein includes a signal peptide of 20-23 amino acids as in human TGF beta 1 and 2, and a precursor protein consisting of 412 amino acids, which can be cleaved at a lys-arg site to produce a 112 amino acid processed peptide containing nine cysteine residues in the same positions as in human TGF beta 1 and 2. At the nucleotide level, the processed coding region of TGF beta 3 shows 72% and 76% identity with the processed coding regions of human TGF beta 1 and TGF beta 2, respectively; at the amino acid level, TGF beta 3 shows 76% identity with TGF beta 1 and 79% identity with TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.     

Complementary DNA cloning of the murine transforming growth factor-beta 3 (TGF beta 3) precursor and the comparative expression of TGF beta 3 and TGF beta 1 messenger RNA in murine embryos and adult tissues

Murine transforming growth factor-beta 3 (TGF beta 3) cDNAs were isolated from a TGF beta 2-induced AKR-2B cDNA library. The composite cDNA sequence is 2894 nucleotides long, including 610-nucleotide and 1054-nucleotide 5' and 3' untranslated sequences, respectively. The murine TGF beta 3-coding region is 1230 nucleotides in length and encodes a precursor protein of 410 amino acids, with a 96% peptide sequence identity with the human TGF beta 3 precursor. Examination of TGF beta 1 and TGF beta 3 mRNA levels in adult murine tissues showed that TGF beta 1 mRNA expression is predominant in spleen, lung, and placenta. In contrast, TGF beta 3 RNA was present in substantial amounts in brain, heart, adipose tissue, and testis. TGF beta 3 mRNA is also observed in adult mouse lung and placenta. Both TGF beta 1 and TGF beta 3 RNAs were present in all stages of mouse fetal development studied from 10.5-17.5 days postcoitum, with higher levels observed in the latter stages. The differential expression of these TGF beta genes suggests that the various TGF beta species may have distinct physiological roles in vivo.     

草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1

A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1-LAP are required for association with mature TGF beta 1.     

Molecular characterization, expression, and functional analysis of two thioredoxins in the black rockfish (Sebastes schlegelii)

Thioredoxins (TRxs) are a family of small evolutionarily conserved proteins that are essential for the maintenance of cellular homeostasis. Two TRx homologue cDNAs were isolated from a black rockfish concanavalin A (Con A)/phorbol myristate acetate (PMA)-stimulated leucocyte cDNA library and named BrTPx1-1 and BrTPx1-2. As compared with other known TRx peptide sequences, the most conserved regions of both BrTRx1-1 and BrTRx1-2 peptides were found to be the redox-active site Trp-Cys-X-X-Cys (WCXXC). The TRx present in most species is a TRx1-2 protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in the larger 13 kDa BrTRx1-1 protein, a Cys-Pro-Pro-Cys (CPPC) active site was identified. Here, we report the identification of a new member of the TRx protein family from the teleost black rockfish, which defines a new subclass of 13-kDa TRx1-1 proteins. Phylogenetic analysis indicated that both BrTRx1-1 and BrTRx1-2 were grouped with other vertebrate TRx1 peptides. BrTRx1-1 expression was strongly induced in peripheral blood leucocytes (PBLs) 12-24 h following Con A/PMA stimulation, with peak expression at 24 h post-stimulation. BrTRx1-2 was induced in PBLs after stimulation with lipopolysaccharide (LPS), Con A/PMA, or poly I:C at 24 h. The BrTRx1-1 gene was predominantly expressed in the liver and gills, while BrTRx1-2 was expressed in PBLs and gills. After treatment with a high concentration (10 μg/mL) of rBrTRx1-1 or rBrTRx1-2, kidney leucocytes exhibited increased cell proliferation and viability under oxidative stress.