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.     

草鱼CD81基因的克隆及功能分析

为研究白细胞表面分化抗原81(CD81)的功能, 对草鱼CD81进行了克隆, CD81全长共1376 bp, 其中5'非翻译区87 bp, 3'非翻译区581 bp, 开放阅读框为708 bp, 包括8个外显子, 7个内含子, 编码235个氨基酸。实验采用实时荧光定量PCR的方法检测了CD81在健康草鱼不同组织中的表达情况及草鱼出血病病毒(GCRV)攻毒前后的表达变化情况。结果显示草鱼CD81在所有被检测组织中均有表达, 在头肾中表达量最高。在GCRV攻毒前后草鱼鳃、脾、肝、肠及头肾5个组织中的CD81表达量均有明显变化。同时, 采用绿色荧光蛋白(GFP)来示踪CD81的亚细胞表达部位, 激光共聚焦显微镜显示, 同人类一样, 草鱼CD81定位于细胞膜上。       

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.     

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.     

黄颡鱼PGRP-L基因克隆及其对爱德华氏菌的反应

为了研究肽聚糖识别蛋白家族(Peptidoglycan recognition proteins, PGRPs)在黄颡鱼(Pelteobagrusfulvidraco)先天免疫应答中发挥的作用, 根据NCBI中斑马鱼(Danio rerio) 和虹鳟(Oncorhynchus mykiss) PGRP-L的基因信息, 采用简并引物和RACE方法从黄颡鱼肝脏中克隆得到了一个长型PGRP (PfPGRP-L)基因. PfPGRP-L基因的全长cDNA序列大小为1617 bp, 其中5'和3'非翻译区的长度分别为135和72 bp, 开放阅读框为1410 bp, 编码469个氨基酸. 同源性和系统进化分析表明, 黄颡鱼PGRP-L与虹鳟的同源性为60%, 与脊椎动物的PGLYRP2 或PGRP-L聚在一起. 半定量RT-PCR分析发现PfPGRP-L基因在黄颡鱼鳃、胸腺、肝脏、脾脏、肠道、肾脏、头肾、心脏、血液和肌肉组织中均有分布, 但在肠道和脾脏中的表达量较为丰富, 而在肌肉和血液中表达则很少. 用爱德华氏菌刺激后, PfPGRP-L在肝脏、脾脏、肠道及头肾中的表达明显上调. 结果表明, PfPGRP-L在黄颡鱼抵抗病原菌中具有重要作用.     

牛ACAD8基因结构及其多态性与生产性状的关联分析

乙酰辅酶A脱氢酶(ACAD)是由核基因编码存在于线粒体中黄色酶的一个家族,它催化脂肪酸的α和β-氧化,该家族第八成员负责催化缬氨酸分解代谢。本研究中获得了牛的ACAD8基因的mRNA和基因组DNA序列并通过基因组DNA与mRNA的比对确定了其基因结构。其mRNA序列包含一个1,251 bp的开放阅读框,一个37 bp 的5′非翻译区和一个 444 bp 的 3′非翻译区;其基因组DNA全长13,814 bp,包含11个外显子和10个内含子。我们从样本中随机选取6个个体进行扩增测序,结果表明牛的ACAD8基因13,408位(GenBank登录号:DQ435445)一个A-G单核苷酸多态。运用PCR-RFLP对不同的基因型进行了区分。对该SNP与来自5个品种共178头无亲缘关系的牛的生产性状进行了关联分析,结果表明:该A-G单核苷酸多态对日增重和嫩度有显著影响(P < 0.05)。具有G等位基因的牛比具有A等位基因的牛生长较快,其肉质也较嫩。因此,牛ACAD8基因在该位点的单核苷酸多态可以作为提高生长速度和牛肉嫩度的标记。     

Ghrelin gene in cichlid fish is modulated by sex and development

We have identified the complementary and genomic DNA sequences of the novel growth hormone-releasing peptide, ghrelin, in the cichlid fish (tilapia Oreochromis niloticus). The tilapia ghrelin precursor cDNA was 855 bp long, consisting of 119 bp of 5(')-untranslated region, 324 bp open reading frame, and 412 bp of 3(')-untranslated region. The serine residues at positions 2 and 3 of the "active core" (GSSF) of the ghrelin mature peptide are conserved between tilapia and the mammalian species. The tilapia ghrelin gene has four exons and three introns and resembles the structure of other known ghrelin genes. RT-PCR analysis revealed ghrelin mRNA predominantly expressed in the stomach but absent in the brain, pituitary, heart, kidney, ovary, and testis. Real-time PCR analysis showed an age/body size dependent increase in gastric ghrelin, which stagnated at 7 cm body size (onset of maturation). Ghrelin mRNA levels were unchanged in food-deprived sexually mature animals but were significantly higher in females compared to males. The present study shows that the structure of ghrelin peptide is highly conserved, and the reported differences in somatic and gonadal growth in tilapia could be a consequence of age- and sex-related synthesis of gastric ghrelin.     

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.     

Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis

Interleukin-21 (IL-21) is an important immune cytokine that was well characterized in human and mammals, but little is known in fish. In present study, an IL-21 homologue was cloned and well characterized from Tetraodon nigroviridis. The full-length Tetraodon IL-21 cDNA was 849bp in size, containing an open reading frame (ORF) of 438bp that translated a 145 amino-acid peptide, a 5' untranslated region (UTR) of 69bp, and a 3' UTR of 342bp. The deduced peptide shared identity of 20-49% with other known IL-21 sequences. The Tetraodon IL-21 gene had six exons while both human and Takifugu IL-21 gene contained only five exons. However, the level of synteny between human, Takifugu and Tetraodon genomes was well conserved during evolution. In vivo expression study showed that Tetraodon IL-21 mRNAs were constitutively expressed at a low level and only in limited tissues, including gut, gill and gonad in healthy fish, and stimulation with LPS increased the expression of IL-21 in these tissues and induced the expression of IL-21 in kidney, spleen and skin, indicating that IL-21 is an inflammatory stress inducible gene associated with the anti-bacterial defense in fish. Our study provided further evidence for the existence of IL-21 in fish, and gained further insight into the immunological functions of IL-21 gene in fish.