Lysozyme Gene Research Articles

Molecular cloning and characterisation of serum lysozyme gene in Muzaffarnagri breed of sheep

The present study was carried out to characterise the serum lysozyme gene in Muzaffarnagri sheep. Total RNA was extracted from the white blood cells and serum lysozyme cDNA was synthesised by Reverse transcription-polymerase chain reaction, then cloned using pGEMT-cloning vector and sequenced. The sequencing of cDNA revealed that the length of serum lysozyme gene was 447 bp with 46% Guanine and Cytosine (GC) content. The comparative sequence analysis of serum lysozyme gene of cattle and buffalo with sheep revealed differences at 24 and 21 places, respectively. The molecular weight of serum lysozyme of sheep is 16.48 kDa. The sheep serum lysozyme cDNA sequence was 94.6% similar to cattle gene sequence, whereas it was 95.3% similar to buffalo gene sequence. Similarly, the derived amino acid sequence of sheep cDNA showed 90.6% similarity with cattle and 92.6% similarity with buffalo. Phylogenetic analysis revealed that sheep and buffalo sequences formed a cluster and evolutionary closer than the cattle sequences.

Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides

Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and “GSTDYGIFQINS” motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His+, Mut+) with multiple hly copies. One transformant resistant to 4.0 mg/ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol.

A goose-type lysozyme gene in Japanese scallop (Mizuhopecten yessoensis): cDNA cloning, mRNA expression and promoter sequence analysis

Lysozyme is an important component of the immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. We constructed a high-quality cDNA library from mantle tissue of adult Japanese scallop (Mizuhopecten yessoensis). The EST which is high homology with g-type lysozyme genes of other species was found in the cDNA library. In the present study, the complete express sequence of g-type lysozyme genes from Japanese scallop (designated as MyLysoG) was directly obtained by PCR. The complete sequence of MyLysoG cDNA consisted of a 5' untranslated region (UTR) of 25 bp, an open reading frame (ORF) of 606 bp, and a 3' UTR of 100 bp with one polyadenylation signal (AATAAA). The deduced amino acids of MyLysoG were 201 amino acids with a putative signal peptide of 18 amino acid residues. It shared the sequence similarity and the common structure features with the g-type lysozyme from other species. Quantitative reverse trancriptase real-time PCR (qRT-PCR) assay demonstrated that mRNA transcripts of g-type lysozyme could be detected in various tissues of unchallenged scallop, and the highest expression of MyLysoG was detected in hepatopancreas tissue. The temporal expression of MyLysoG in hemolymph after Vibrio anguillarum challenge was up-regulated and reached the maximum level at 3h post stimulation, and then dropped back to the original level even lower than the control group. Furthermore, a 978 bp of 5'-flanking sequence of MyLysoG was identified by genome walking, and several potential transcription factor binding sites (TFBS) were detected in the putative promoter region. One part of the MyLysoG promoter region contains nine sites of SNPs and three sites of insert-deletion (indel) polymorphisms, and these mutations were found organize into two haplotypes. The two haplotypes were associated with different TFBS. The haplotypes could be selected to analyze the transcriptional-level control of scallop g-type lysozyme gene and the scallop immune system.

Identification and expression analysis of goose-type lysozyme in half-smooth tongue sole (Cynoglossus semilaevis)

Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5′-terminal untranslated region (UTR), a 452 bp 3′-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5′-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h post-infection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection.

Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded.

Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli.

Expression of Lysozymes from Erwinia amylovora Phages and Erwinia Genomes and Inhibition by a Bacterial Protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned ‘silent’ lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.

Targeting the human lysozyme gene on bovine αs1-casein gene locus in fibroblasts

Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the expression of human lysozyme could be regulated by the endogenous cis-element of αs1- casein gene in bovine mammary glands. In order to analyze the bioactivity of the vector, the targeting vector was stably transfected and randomly integrated into mouse mammary epithelial cells. Reversetranscription- polymerase chain reaction (RT-PCR) and western blot results showed that, the bovine αs1-casein promoter in the 5’ arm was able to direct the efficient expression and secretion of human lysozyme in mammary epithelial cells. Turbidimetric assay showed that the antibacterial activity of lysozyme in transfected cells culture medium was 180 U/ml. To obtain the gene targeted cells line, bovine fetal fibroblasts were isolated and transfected with linear targeting vector (21.9 kb) using nucleofector device, which the transfection rate was about 25%. After seven rounds of independent cell transfection, a total of 8 × 10 7 cells were transfected, 118 colonies were expanded and analyzed by PCR, but none were found to be targeted. However, the targeted events were detected in the mixed cells which did not formed obvious colonies in five 10 cm dishes. Thus, these results indicate that, the 8.2 kb exogenous genes could be site specifically integrated into the transcriptionally silent αs1-casein gene locus in fibroblasts, but the unfavorable chromatin structure in such loci may have a disadvantage to targeted colonies formation in expansion stage. We suggest that minimizing the length of in vitro culture time and relax selection as soon as colonies become evident might prevent such loss of targeted cells. Key words: Gene targeting, human lysozyme, bovine αs1-casein, C127 cells, fetal fibroblasts.

Protist-type lysozymes of the nematode Caenorhabditis elegans contribute to resistance against pathogenic Bacillus thuringiensis.

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens.

Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins

Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase ( P < 0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host–parasite, i.e. snail–trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail–trematode system.

Characterization and Application of Oviductal Epithelial Cells In Vitro in Gallus domesticus1

Chicken oviductal epithelium produces large quantities of egg white protein in daily cycles. In this study, we cultured and characterized oviductal epithelial cells (OECs) from juvenile (10-wk-old) chickens and from actively laying (30-wk-old) hens. The juvenile OECs were maintained over passage 25 and were positive for toluidine blue, lectin-ConA, HPA, UEA-1, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR, whereas the adult OECs were cultured over passage 6 and were positive for toluidine blue, periodic acid-Schiff, lectin-ConA, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR. To investigate the optimal concentration of steroid hormones for inducing egg white protein genes in vitro, we examined the effects of estrogen, diethylstilbestrol, progesterone, and corticosterone on OECs. Results showed that oviduct-specific levels of avidin, ovalbumin, ovomucin, lysozyme, ESR1, and PGR gene expression were significantly elevated in steroid hormone-treated OECs compared with those of untreated cells (P < 0.05). Ovalbumin protein was also secreted into culture medium from hormone-treated OECs. In addition, to examine the application of OECs for avian transgenesis, we introduced human thrombopoietin (THPO)-expressing lentiviral vector controlled by a 3.5-kb ovalbumin promoter into cultured OECs, and THPO expression was significantly induced with diethylstilbestrol or progesterone in juvenile OECs (P < 0.05) and in adult OECs (P < 0.05). In conclusion, these data demonstrate the potential of cultured OECs as a model system for providing a better understanding of the regulation of gene expression and for the production of an avian transgenic bioreactor.

Selection for improved resistance to Aeromonas hydrophila in Indian major carp Labeo rohita: Survival and innate immune responses in first generation of resistant and susceptible lines

Selection for disease resistance in fish may be performed directly on basis of survival data obtained in controlled challenge trials, or indirectly using information from immunological or molecular markers linked to differential survival. In the present study, several key innate immune parameters were measured in aeromoniasis resistant and susceptible lines of rohu Labeo rohita to assess their suitability as immune markers for use in indirect selection for increased resistance. Experimental infection with Aeromonas hydrophila (9.55 × 10 6 cfu g −1 fish) through the intraperitoneal route produced higher survival in the resistant line (73.33%) as compared to the susceptible line (16.67%). Blood and liver tissue samples from both lines were collected to study some of the innate immune parameters and immune-related gene expression. The respiratory burst activity of blood phagocytes, serum myeloperoxidase activity and ceruloplasmin level were significantly ( p < 0.05) higher in the resistant line compared to the susceptible line. Lower level of blood glucose and serum natural haemolysin titre were marked in the resistant line as compared to the susceptible line. No significant difference was measured in total serum protein concentration, antiprotease activity and bacterial agglutinin level between two lines, while the expression of transferrin, complement factor C3 and TLR 22-like transcripts were significantly ( P < 0.05) higher in liver samples of the susceptible line. However, no such difference was found in β 2-microglobulin and lysozyme gene expression between lines. The study demonstrated the possibility of using some of the investigated innate immune parameters as indirect marker traits for selection for improved resistance to aeromoniasis in rohu.

Evolution of the mammalian lysozyme gene family.

BackgroundLysozyme c (chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known.ResultsHere we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution.ConclusionsThe mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.

Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16

Lysozyme is an enzyme that is essential for protection against bacterial infections. In this study, a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H. polymorpha chromosome. Recombinant T4 lysozyme was successfully expressed in the yeast H. polymorpha A16; 0.49 g L(-1) secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0. Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria, Micrococcus lysodeikticus, and the gram negative bacteria Xanthomonas campestris pv. malvacearum and Xanthomonas oryzae pv. oryzae. The zone of inhibition assay was used to evaluate antimicrobial activity. Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme. SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme. SDS-PAGE without 0.2 mol L(-1) dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed inter- and intra-molecular disulfide bonds which resulted in loss of enzyme activity.

INTERACTION OF VIRAL AND BACTERIAL LYSOZYMES WITH ERWINIA AMYLOVORA AND THEIR INHIBITION BY A BACTERIAL PROTEIN

Viral lysozymes can interact with Erwinia amylovora, and their genes may control fire blight when expressed in the pathogen. We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species. With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes. Fusion proteins with the (phi)Ea1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly. For large fusions at the N- or C-terminus lytic activity was completely lost. When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored. Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme. Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with (phi)Ea1h lysozyme and T4 lysozyme. The cellular expression of a viral lysozyme gene can be toxic for a bacterial host. The gene was therefore expressed in yeast as safe environment for its secretion. Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast. When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen. The (phi)Ea1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage (phi)Ea104 and (phi)Ea116 was not affected by the enzyme inhibitor.

Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris

Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS–PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni–NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M. lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.

Effects of environmental salinity, biopsy, and GH and IGF-I administration on the expression of immune and osmoregulatory genes in the gills of Atlantic salmon (Salmo salar)

Immune-endocrine interactions are thought to be important for fish health, especially following injury of peripheral tissues. We investigated the influences of gill biopsy (surgical removal of several gill filaments with minimal bleeding) on mRNA levels of osmoregulatory and immune genes in peripheral tissues of Atlantic salmon (Salmo salar) smolts in freshwater (FW) and seawater (SW). The effects of growth hormone (GH) and insulin-like growth factor (IGF)-I, which are known as both osmoregulatory and immunomodulatory hormones, were also examined in FW- and SW-acclimated fish. Compared with the intact gill arch of the same fish, the residuary filaments after biopsy showed significant reduction in Na+,K+-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels in FW fish. In contrast, mRNA levels of these genes in the filaments with biopsy were increased in SW fish. Increased caspase gene transcription was observed in the cut filaments of SW fish, but not in those of FW fish. Lysozyme gene transcription was stimulated in the residuary gill filaments after biopsy in both FW and SW. Administration of salmon GH or IGF-I increased mRNA levels of NKA, CFTR, caspase, and lysozyme in the gills after biopsy in FW fish. In SW-acclimated fish, there was no significant effect of GH or IGF-I on the transcription of osmoregulatory or immune genes. The results in this study showed tight relationships between immunity, osmoregulation, and endocrine system in fish gills, and that nonlethal gill biopsy can be used for investigations of fish health.

Influence of insulators on transgene expression from integrating and non-integrating lentiviral vectors

BackgroundThe efficacy and biosafety of lentiviral gene transfer is influenced by the design of the vector. To this end, properties of lentiviral vectors can be modified by using cis-acting elements such as the modification of the U3 region of the LTR, the incorporation of the central flap (cPPT-CTS) element, or post-transcriptional regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE). Recently, several studies evaluated the influence of the incorporation of insulators into the integrating lentiviral vector genome on transgene expression level and position effects.MethodsIn the present study, the influence of the matrix attachment region (MAR) of the mouse immunoglobulin-κ (Ig-κ) or the chicken lysozyme (ChL) gene was studied on three types of HIV-1-derived lentiviral vectors: self-inactivating (SIN) lentiviral vectors (LV), double-copy lentiviral vectors (DC) and non-integrating lentiviral vectors (NILVs) in different cell types: HeLa, HEK293T, NIH-3T3, Raji, and T Jurkat cell lines and primary neural progenitors.Results and DiscussionOur results demonstrate that the Ig-κ MAR in the context of LV slightly increases transduction efficiency only in Hela, NIH-3T3 and Jurkat cells. In the context of double-copy lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency. In the same way, in the context of non-integrating lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency, except in differentiated primary neural progenitor cells.The ChL MAR in the context of integrating and non-integrating lentiviral vectors shows no effect or a decrease of transgene expression in all tested conditions.ConclusionsThis study demonstrates that MAR sequences not necessarily increase transgene expression and that the effect of these sequences is probably context dependent and/or vector dependent. Thus, this study highlights the importance to consider a MAR sequence in a given context. Moreover, other recent reports pointed out the potential effects of random integration of insulators on the expression level of endogenous genes. Taken together, these results show that the use of an insulator in a vector for gene therapy must be well assessed in the particular therapeutic context that it will be used for, and must be balanced with its potential genotoxic effects.

Characterization of six antibacterial response genes from the European corn borer (Ostrinia nubilalis) larval gut and their expression in response to bacterial challenge

Six cDNAs encoding putative antibacterial response proteins were identified and characterized from the larval gut of the European corn borer (Ostrinia nubilalis). These antibacterial response proteins include four peptidoglycan recognition proteins (PGRPs), one β-1,3-glucanase-1 (βglu-1), and one lysozyme. Tissue-specific expression analysis showed that these genes were highly expressed in the midgut, except for lysozyme. Analysis of expression of these genes in different developmental stage showed that they were expressed in larval stages, but little or no detectable expression was found in egg, pupa and adult. When larvae were challenged with Gram-negative bacteria (Enterobacter aerogenes), the expression of all six genes was up-regulated in the fatbodies. However, when larvae were challenged with Gram-positive bacteria (Micrococcus luteus), only PGRP-C and lysozyme genes were up-regulated. This study provides additional insights into the expression of antibacterial response genes in O. nubilalis larvae and helps us better understand the immune defense response in O. nubilalis.