[Show abstract][Hide abstract] ABSTRACT: The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones. In this study, a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified. The tHsc70 full-length complementary DNA (cDNA) is 2272 bp long with a 1941-bp open reading frame (ORF) encoding 646 amino acids. Three characteristic signature regions of the HSP70 family, two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD), and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence. The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting. Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level, and 49.1% to 99.5% at the amino acid level with the known Hsc70s. Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis). The tHsc70 was expressed in the liver, lung, heart, and skeletal muscle. The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C. Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress. Irrespective of different profiles of expression under heat stress, tHsc70 may play roles in protecting turtles from thermal stress.
Journal of Zhejiang University SCIENCE B 06/2012; 13(6):465-77. DOI:10.1631/jzus.B1100309 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chinese soft-shelled turtles (Trionyx sinens) in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5~80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA). Detailed morphological examinations were also conducted to confirm the white-spot disease.
Journal of Zhejiang University SCIENCE B 08/2008; 9(7):578-81. DOI:10.1631/jzus.B0720009 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands's isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.
Journal of Zhejiang University SCIENCE B 03/2008; 9(2):148-53. DOI:10.1631/jzus.B0710395 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genus Listeria consists of six species: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri and L. grayi. Two of the species, L. monocytogenes and L. ivanovii are pathogenic. The heterogeneity of remaining species, previously assumed to be nonpathogenic, regarding their capability of acquiring virulence-associated genes may reflect their potential ability to be causative agents of diseases, especially in immunocompromised mannals. Virulence determinants involved in environmental tolerance, adhesion and invasion of eukaryotic cells and intracellular life function interactively. The virulence genes are mostly organized into discrete genetic units known as pathogenicity islands (PAIs), among which Listeria pathogenicity island 1 (LIPI-1) and island 2 (LIPI-2) are the most important. During the evolution of pathogenicity, a common ancestor bearing PAIs gave rise to the currently prevailing typical strains of six species through horizontal transfer of virulence determinants or by events such as recombination and natural selection. Bacteriophages, transposons and plasmids might play critical roles in these processes as the executants. Compred to pathogenic species, the nonpathogenic species lost LIPI-1 (L. innocua, L. welshimeri and L. grayi) or harbored corrupted LIPI-1 (L. innocua, L. welshimeri). Some types of natural atypical Listeria strains such as nonhemolytic L. seeligeri and hemolytic L. innocua, although complicating taxonomic identification, should contribute fruitful insights into the evolution events of pathogenicity underlying the phylogeny of the genus Listeria.
[Show abstract][Hide abstract] ABSTRACT: Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus, it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections,and its pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.
Journal of Zhejiang University SCIENCE B 04/2007; 8(3):162-9. DOI:10.1631/jzus.2007.B0162 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.
Journal of Zhejiang University SCIENCE B 12/2006; 7(11):922-8. DOI:10.1631/jzus.2006.B0922 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P < 0.05). Such insertional mutation resulted in reduced virulence, about 3.7 logs and 6.5 logs less than its parent strain L. monocytogenes 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models respectively. The recombinant strain was relatively stable as shown by amplification of the target gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.
[Show abstract][Hide abstract] ABSTRACT: A low-pathogenicity isolate of Listeria monocytogenes from cow's milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes (iap, prfA, plcA, hly, mpl, actA, plcB, InlA and InlB) were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models (50% lethal dose: 10(8.14) vs. 10(5.49) and 10(6.73) vs. 10(1.9), respectively). The genes prfA, plcA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD (>98%). Genes iap, hly, plcB, InlA and InlB of L. monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.
[Show abstract][Hide abstract] ABSTRACT: Effects of plasmid type and insertion sequence on in vitro and in vivo multiplication and invasiveness of attenuated Salmonella typhimurium were examined following transformation of the bacteria with eukaryotic expression plasmids pcDNA3 and pCI with or without heterologous gene (Newcastle disease virus F gene). Exogenous plasmids had negative impacts on the replication or invasiveness of the attenuated S. typhimurium in LB broth and/or HeLa cell monolayers as well as on its survival in live chicks. The plasmid pCI had more significant effects than pcDNA3. Introduction of heterologous gene into the plasmids not only exhibited additional negative influence on the host strain but also on their own stability therein. All these results suggest that full consideration should be given to the types of plasmids and their stability within the host strain as well as to their effects on replication and invasiveness of attenuated bacteria as the DNA vaccine delivery vector for improved immune protection.
[Show abstract][Hide abstract] ABSTRACT: In this study, the haemolytic activities of Astragalus membranaceus saponins (AMS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against OVA were evaluated. We determined the haemolytic activity of AMS using 0.5% rabbit red blood cell. AMS showed a slight haemolytic effect, with its haemolytic percent being 0.66% at the concentration of 500 microg/ml. Furthermore, the adjuvant potentials of AMS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were investigated. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg) or AMS (50, 100 or 200 microg) on Day 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. AMS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.05 or P<0.001). OVA-specific IgG, IgG1 and IgG2b antibody titers in serum were also significantly enhanced by AMS compared with OVA control group (P<0.01 or P<0.001). Moreover, no significant differences (P>0.05) were observed between enhancing effect of AMS and QuilA on the OVA-specific IgG, IgG1 and IgG2b antibody responses to OVA in mice. In conclusion, the results suggest that AMS could be safely used as adjuvant with low or non-haemolytic effect.
[Show abstract][Hide abstract] ABSTRACT: To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogenes could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.
[Show abstract][Hide abstract] ABSTRACT: To examine if polyprotein gene (VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam- and phoP-), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2004; 20(3):437-40.
[Show abstract][Hide abstract] ABSTRACT: Mice and 3-day-old chickens were orally inoculated with the recombinant attenuated Salmonella typhimurium strain ZJ111 carrying pcDNA3-F expression plasmid encoding the fusion protein of Newcastle disease virus (NDV). The results showed that ZJ111/pcDNA3-F was relatively safe. The recombinant plasmid pcDNA3-F was stable within the host stain ZJ111 in vitro and in vivo as shown by restriction enzyme analysis and PCR identification of the F gene. In an experimental vaccination study, 3-day-old chickens were orally immunized with ZJ111/pcDNA3-F with a dose of 108 cfu per chicken and boosted two weeks later. At week 4 post boosting, all chickens were challenged with a lethal dose of a virulent NDV strain F48 E9. The results showed that oral vaccination with ZJ111/pcDNA3-F induced stronger humoral and cellular immune responses than intramuscular immunization with naked pcDNA3-F plasmid. It also exhibited higher protection rate than the latter (66.7% vs 50%). This study indicates that the DNA vaccine using attenuated Salmonella typhimurium as delivery carrier had good safety, stability and immunogenicity and exhibited good potential of low cost and convenience for poultry disease control.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2003; 19(1):24-9.
[Show abstract][Hide abstract] ABSTRACT: The full-length cDNA of fusion protein (F) gene of newcastle disease virus (NDV)strain F48E9 was amplified by RT-PCR and inserted into pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. The resulting recombinant plasmid pcDNA3-F was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam(-) and phoP(-)), which was then used to transfect the Vero cells. The DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells. There was expression of the F protein as shown by indirect immunofluorescent assay. The expression began at 48 h post-infection and increased thereafter, as indicated by ELISA. A 55 kD band of the F protein was identified by SDS-PAGE and Western blotting. These results clearly show that the expressed fusion protein was immuno-reactive with chicken anti-NDV serum.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 08/2002; 34(4):488-93.