Uptake of channel catfish virus from water by channel catfish and bluegills

Radiolabeled channel catfish virus entered the gills of juvenile channel catfish (Ictalurus punctatus) and was then concentrated in the gut and the liver over 48 hours. Diminution of radioactivity was not detected in these tissues over the course of the experiment. Bluegills (Lepomis macrochirus) were capable of clearing the virus during a period of 48 hours. [3H]Thymidine alone had a different distribution in the channel catfish than did labeled virus.     

Molecular and functional characterization of channel catfish (Ictalurus punctatus) neutrophil collagenase

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.     

猪ADSL基因克隆及其在部分组织中的mRNA定量表达

试验以通城猪为研究对象,在对猪ADSL基因全长编码区cDNA片段进行克隆、序列测定、与GenBank中已收录的其他14种动物的ADSL全长编码区序列进行同源性分析,并构建系统发生树的基础上,采用实时(相对)定量PCR方法对刚出生和65日龄两个不同生长发育时期的通城猪心、肝、肾脏、背最长肌4种组织的ADSL基因表达谱进行分析。结果表明,所克隆的ADSL基因片段大小为1 548 bp(GenBank登录号:GU249574),其中包含一长为1 455 bp的完整阅读框,通过核酸序列分析发现,该基因编码484个氨基酸;与GenBank中已收录的猪ADSL基因序列同源性最高,达99.9%;与恒河猴和黑猩猩的序列同源性最低,分别为66.3%和41.3%;15种动物的20条ADSL全长CDS序列聚类分布为两个主支。刚出生时的通城猪ADSL基因在心、肝、肾脏组织的mRNA表达水平均高于65日龄通城猪的心、肝、肾脏组织中的mRNA表达;然而,其背最长肌的ADSL基因mRNA表达水平则低于65日龄的通城猪。本结果为猪ADSL基因的生物学功能研究和遗传进化研究提供了分子依据。     

Molecular cloning of serogroup- and serotype-specific genome segments from bluetongue virus serotype 11

Genome segments 2, 6, 8, and 9 of bluetongue virus (BTV) serotype 11, coding for P2, NS1, NS2, and P6, respectively, were cloned into pUC 8. Sizes of segment-2 and segment-6 clones indicated partial copies (55% and 80% of full length, respectively), whereas segment 8 and 9 clones represented full-length copies. Northern blot hybridizations of the clones to the 5 United States BTV prototypic serotypes (2, 10, 11, 13, and 17) revealed segment-2 clone to be serotype-specific to BTV-11, whereas segment 6, 8, and 9 clones were able to detect all serotypes to varying degrees. All clones failed to detect the related orbivirus, epizootic hemorrhagic disease virus.     

Molecular cloning of the SMAD4 gene and its mRNA expression analysis in ovarian follicles of the Yangzhou goose (Anser cygnoides)

1. Mothers against decapentaplegic homolog 4 (SMAD4) is an important protein in animal reproduction. It plays pivotal roles in cellular pathways, including apoptosis. The expression profile of the SMAD4 gene in goose ovarian follicles has not been reported.

2. In this study, the SMAD4 coding sequence was cloned from the Yangzhou goose. A phylogenetic analysis was performed and mRNA expression was examined in various tissues using quantitative real-time PCR.

3. An alternative splice form of SMAD4, SMAD4-b having 1656 bp, was identified. SMAD4-a mRNA was widely expressed in various healthy tissues, whereas SMAD4-b was very weakly expressed. SMAD4 mRNA in the ovary and oviduct was significantly higher than that in the pituitary and hypothalamus. SMAD4 mRNA expression analysis in hierarchical follicles showed that the level of SMAD4 mRNA was higher in large white follicles and post-ovulatory follicles than in the other follicles.

4. The results indicate that SMAD4 might be involved in the recruitment of hierarchical follicles.

     

Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris)

Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish (Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5' and 3' ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5'-terminal untranslated region (UTR), a 520 bp 3'-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58-75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions (d(N)) occurred at a higher frequency than that of synonymous substitutions (d(S)), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3'-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and stomach, and low or negligible expression in heart. Homology modelling has been applied to create a 3D model of longsnout catfish MHC class IIB, with features evaluated and discussed to investigate its interaction with CD4 participating in antigen recognition. The present findings will provide valuable information for understanding structure, function, expression, and molecular polymorphism of MHC class IIB in adaptive immunity of the Chinese longsnout catfish and teleost.     

Molecular cloning and expression of bottlenose dolphin (Tursiops truncatus) interleukin-8

The bottlenose dolphin interleukin (IL)-8 cDNA was molecularly cloned. The dolphin IL-8 has an open reading frame of 303-bp encoding 101 amino acids. The homology of the amino acid sequence with that of other species was: sheep, 89.1%; cattle, 88.1%; pig, 85.1%; dog, 85.1%; horse, 79.2%; human, 74.5%; and macaque, 72.3%. The amino acid sequence suggested that dolphin IL-8 was a CXC chemokine. The recombinant dolphin IL-8 protein was recognized with anti-ovine IL-8 monoclonal antibody.     

Molecular cloning and expression analysis of porcine gamma-interferon-inducible lysosomal thiol reductase (GILT)

A porcine interferon-gamma-inducible lysosomal thiol reductase (GILT) cDNA, designated pGILT, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pGILT consists of 1,062 bp with a 741 bp open reading frame, encoding 246 amino acids, with a putative molecular weight of 29.5 kDa. The deduced pGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The genomic DNA sequence of pGILT contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. The result of real-time PCR showed that GILT is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, blood and kidney. And the pGILT expression is obviously up-regulated in spleen and blood after induction with LPS. These results suggesting that pGILT is highly likely to play a role in the innate immune responses in porcine. It also provided the basis for investigations on the role of GILT in this important domestic species and an animal model for human diseases.     

Pharmacokinetics of gentamicin in channel catfish (Ictalurus punctatus)

The pharmacokinetics of gentamicin were determined in male and female Ictalurus punctatus weighing between 0.3 kg and 1.7 kg. Plasma gentamicin concentrations were measured by radioimmunoassay technique. In the 1st experiment, an intracardiac bolus dosage of gentamicin (1 mg/kg) was given to 10 channel catfish. Samples of blood were obtained (by cardiac puncture) immediately before gentamicin was given and at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0003 minute-1). The maintenance therapeutic IV dosage of gentamicin was calculated to be 1.6 mg/kg given at 33.2-hour intervals. In the 2nd experiment, an IM dosage of the drug (1 mg/kg) was given to 9 channel catfish, and samples of blood were obtained at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0002 minutes-1). The maintenance therapeutic IM dosage of gentamicin was calculated to be 3.5 mg/kg given at 33.2-hour intervals. The IM bioavailability of gentamicin in channel catfish was estimated to be 60%. Other pharmacokinetic values were also determined. It was concluded that the therapeutic regimen of choice for gentamicin in channel catfish was 3.5 mg/kg given IM with 33-hour intervals between doses.     

山羊CCRL2基因的分子克隆和表达分析

本研究旨在克隆山羊趋化因子(C-C基序)受体2(chemokine(C C motif)receptor-like 2,CCRL2)基因序列,并研究其组织和时序表达谱。以简州大耳羊为试验材料,应用RT-PCR方法克隆CCRL2基因并进行生物信息学分析,qPCR方法构建该基因在山羊不同组织表达谱和不同月龄背最长肌、股二头肌和臂三头肌中的表达水平,并将基因表达与IMF含量进行相关分析。结果显示:克隆得到山羊CCRL2基因(GenBank登录号:KT165378)长度为1 143bp,编码区长度为1 047bp;qPCR分析表明,CCRL2mRNA在24月龄山羊心、肝、脾、肺、肾、脂肪、背最长肌、股二头肌和臂三头肌中均有不同程度的表达,其中脾表达量最高(P<0.01);时序表达中,CCRL2基因在股二头肌和臂三头肌中的表达量均在8~10月龄极显著高于1~3和24月龄(P<0.01),在背最长肌中则为24月龄极显著高于1~3和8~10月龄(P<0.01);相关分析显示,股二头肌中CCRL2基因mRNA表达量与IMF含量呈极显著负相关(P<0.01)。CCRL2基因可能参与山羊IMF沉积作用。     

急性感染期猪伪狂犬病病毒部分基因组染色质状态

为探究急性感染期猪伪狂犬病病毒(PRV)在宿主细胞内基因组染色质的状态,首先建立PRV实时荧光定量PCR(qPCR)方法并测定0.1 MOI PRV感染Neuro-2a细胞后病毒基因组的复制动态,然后收集PRV感染Neuro-2a细胞的样品进行染色质免疫共沉淀试验(CHIP),并通过qPCR测定病毒部分DNA与组蛋白H3结合形成的染色质状态。结果显示,PRV急性感染Neuro-2a细胞后,部分基因组以染色质结构存在,并与病毒复制有一定相关性。本研究成功建立用于研究PRV基因组染色质状态的CHIP试验方法,为PRV急性感染期表观遗传学研究提供依据。     

Effects of recombinant bovine somatotropin on growth and abundance of mRNA for IGF-I and IGF-II in channel catfish (Ictalurus punctatus)

Research was conducted to examine growth rates, circulating concentrations of IGF-I, and mRNA abundance levels of IGF-I and IGF-II in channel catfish (Ictalurus punctatus) given recombinant bovine ST (rbST; Posilac, Monsanto Co., St. Louis MO). In the first study, juvenile catfish (5.5 +/- 0.5 g) were randomly assigned to one of three treatments: 1) sham-injected control (one needle puncture per week); 2) rbST (30 microg x g BW(-1) x wk(-1); Posilac); and 3) nonhandled control (control). At the end of the 6-wk study, the fish were weighed, measured for length, and G:F was determined. Compared with sham and control treatments, rbST-treated fish had 48% greater final BW, 14% greater total length, and 52% greater G:F (P < 0.001). In the second study, juvenile catfish (41.1 +/- 1.5 g) were assigned randomly to one of two treatments: 1) sham or 2) rbST. Eight fish per treatment were sampled on d 0, 1, 2, 7, 14, and 21 for blood, muscle, and liver. Relative expression of IGF-I and IGF-II mRNA was determined by real-time PCR and plasma concentrations of IGF-I were measured using a validated fluoroimmunoassay. Circulating concentrations of IGF-I were increased (37.9 +/- 5.5 vs. 22.0 +/- 6.6 ng/mL; P < 0.05) in rbST-injected fish compared with sham-injected controls by d 14. Liver IGF-I and IGF-II mRNA was increased 4.3-and 14.4-fold, respectively, by d 1 in rbST-injected fish compared with controls (P < 0.05); however, abundance of liver IGF-I and IGF-II mRNA did not differ from controls on d 0, 2, 7, 14, and 21. Abundance of muscle IGF-I and IGF-II mRNA did not differ in rbST-injected fish compared with controls throughout the study. Results of the first study demonstrated that rbST improves growth performance of channel catfish. Results of the second study showed that the growth-promoting effects of rbST were not mediated by the expression of IGF-I or IGF-II mRNA in the muscle. Instead, the results suggest that rbST promotes growth by stimulating plasma IGF-I release, possibly through its direct effect on the liver or on local tissues to synthesize IGF-I. The changes in mRNA abundance and plasma concentrations of IGF-I support the role of IGF-I in growth regulation of channel catfish.     

Molecular cloning of the mRNA coding for the G protein of the viral haemorrhagic septicaemia (VHS) of salmonids

Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming. The development of a recombinant subunit vaccine could solve that problem. The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen. The G protein has a molecular weight of 65 kDa, reduced to 55 kDa by deglycosylation. cDNA was synthetized from mRNA of VHS virus infected cells, and cloned in E. coli. The viral cDNA was recognized by positive hybridization with a labelled probe made from infected cell RNA, and negative hybridization with labelled cDNA made from cellular RNA. The Northern blot hybridization with different clones on VHS infected cell RNA revealed two VHS mRNA whose lengths, 2.0 and 1.5 kb, were compatible with the mRNA length for G and N proteins respectively. This mRNA must contain about 400 bp of untranslated sequence.     

潜伏感染期猪伪狂犬病病毒基因组甲基化预测及图谱测定

为研究DNA甲基化在PRV潜伏感染中的作用,本研究首先对PRV全基因组甲基化状态进行预测,再采用亚硫酸氢盐PCR测序的方法检测急性期及潜伏感染期IE180及EP0启动子区及转录起始位点附近区域的甲基化状态。结果显示,急性期及潜伏感染期IE180及EP0启动子区及转录起始位点附近区域均存在散在的甲基化位点,并未发现广泛的甲基化区域,与预测结果一致。结果表明,潜伏感染期间PRV基因组发生甲基化的可能性很小,进而可知DNA甲基化在PRV潜伏感染过程中未起到关键的作用。