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ABSTRACT: Orf virus (ORFV) is an enveloped virus with a double-stranded DNA genome, causing a contagious pustular dermatitis, mainly in goats and sheep. In this study, two strains of ORFV were isolated from sheep and goat samples in Xinjiang and Shaanxi, China. The B2L and virus interferon resistance (VIR) genes of these two isolates were sequenced and analyzed after PCR amplification. Phylogenetic analysis showed that the two isolates clustered with other ORFV strains but were separated into different subgroups. The Xinjiang strain shared the highest homology with the Gansu strain, whereas the Shaanxi strain shared higher homology with the Taiwan and Hubei strains. This is the first report of the molecular characterization of ORFV in Northwest China, and it provides new information on the genotyping of the causative agents responsible for contagious ecthyma dermatitis outbreaks in China.
Archives of Virology 02/2013; · 2.11 Impact Factor
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ABSTRACT: The goal of this study was to evaluate the specificity of a polyclonal antibody against the F protein from Peste des petits ruminants virus (PPRV). A pET30a/F prokaryotic expression vector was successfully constructed and its recombinant protein was expressed. The result of Western blot analysis showed that the fusion protein pET30a/F possessed good immunoreactivity and the purified recombinant protein was then used as the antigen to raise anti-pET30a/F polyclonal antibody in rabbits. The polyclonal antibody titer against the recombinant F protein was confirmed by indirect ELISA, and the protein's specificity against pET30/F polyclonal antibody was confirmed by both Western blot and indirect immunofluorescence assay in transfected cells. In short, we obtained the high-level expression of recombinant F protein as well as high titers of rabbit polyclonal antibody specificity against F protein in pCAGGS/F transfected cells. This special polyclonal antibody offers a valuable and useful tool for further study of the pathogenesis of PPRV early infection and the structural and functional characterization of PPRV F protein.
Monoclonal antibodies in immunodiagnosis and immunotherapy. 02/2013; 32(1):26-31.
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ABSTRACT: Rabbit cysticercosis, caused by the larval stage of Taenia pisiformis, is a serious parasitic disease of rabbits. It was reported that some cysteine peptidases have potential roles in the pathogenesis of various parasitic infections. To investigate the biochemical characteristics and roles in the pathogenesis/host-invasion of cysteine peptidases, a cDNA sequence encoding for a cathepsin L-like cysteine protease (TpCP) was cloned and identified from the T. pisiformis metacestodes. This sequence was 1220bp in its length, which included a 1017bp open reading frame encoding a 339 amino acid peptide. Multiple sequence alignments revealed a 28.9-88.5% similarity with cathepsin L-like cysteine proteases from other helminth parasites and mammals. The recombinant TpCP expressed in Escherichia coli did not show the proteolytic activity by zymography gel assay. However, the TpCP expressed in Pichia pastoris had typical biochemical activities that could hydrolyze rabbit immunoglobulin G, bovine serum albumin and fibronectin. Substrate studies indicated pronounced cleavage of Z-Phe-Arg-AMC. This activity was sensitive to cysteine protease inhibitor E-64 and immunohistochemistry results also indicated that TpCP was distributed as an intense positive reaction in the bladder wall. Our results gave us insights into future studies of TpCP's roles in the infection.
Veterinary Parasitology 01/2013; · 2.58 Impact Factor
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Juntao Ding,
Yadong Zheng,
Ying Wang,
Yongxi Dou,
Xiaoyu Chen,
Xueliang Zhu,
Shuai Wang,
Shaohua Zhang,
Zhenyong Liu,
Junling Hou,
Junjun Zhai,
Hongbin Yan, Xuenong Luo,
Xuepeng Cai
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ABSTRACT: A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 10(12) CFU of S. typhimurium χ4558, while all mice infected with 10(7)CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4(+) and CD8(+) T lymphocytes and the percentage of helper CD4(+)/CD8(+) T lymphocytes was significantly increased (p<0.01). After oral administration, S. typhimurium χ4558 was first colonized mainly in the Peyer's patches and then predominantly in the mesenteric lymph nodes and spleens in the vaccinated mice. In addition, the high levels of specific anti-TSOL18 antibodies were also observed in pigs administrated with S. typhimurium χ4558. Collectively, these results demonstrate the possibility of use of an attenuated S. typhimurium strain as a vector to deliver protective antigens of T. solium.
Comparative immunology, microbiology and infectious diseases 12/2012; · 2.99 Impact Factor
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ABSTRACT: In 2007, herds of pigs in Jiangxi Province, China experienced outbreaks of a severe form of suspected porcine reproductive and respiratory syndrome (PRRS) characterized by high fever, high morbidity and mortality in animals of different ages. 152 swine sera and 42 tissues (consisting of liver, lung, lymph node and kidney) from five herds of pigs were collected. Pigs were diagnosed as infected with a highly pathogenic form of the PRRS virus (PRRSV) based on ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) results. Serological surveys indicated that 67-100% of the examined pig herds in Jiangxi Province were seropositive. 42 tissue samples were used to detect classical swine fever virus, porcine circovirus type 2 and PRRSV. Results indicated that only PRRSV was detected in 42 samples. 12 PRRSV amplified products of five herds, which consisted of two or three samples randomly selected from each herd, were used for sequencing. Subsequent nucleotide sequencing showed that the NSP2 gene had 99-99.7% nucleotide and 99.2-100% derived amino acid sequence identities among 12 tissues with that of the PRRS-JXA1 strain, deletions of 29 amino acids corresponded to positions 534-562 of the NSP2 gene sequence. These results revealed that the diseased pigs were all caused by fatal PRRSV variant. Compared with the same period in 2006, the number of positive cases from Jiangxi Province remained unchanged. These findings demonstrated that the highly pathogenic Northern American type PRRSV was still spreading in Jiangxi Province, China in 2007.
Irish veterinary journal. 07/2012; 65(1):14.
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ABSTRACT: Ovine β2 subunit of the interleukin (IL)-12 receptor (IL-12Rβ2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rβ2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rβ2. The ovine IL-12 Rβ2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rβ2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rβ2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rβ2 mRNA expression was detected at all the detected tissues with the exception of thymus.
Gene 01/2012; 499(1):124-9. · 2.34 Impact Factor
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ABSTRACT: As the linker between the A chain and B chain of proinsulin, C-peptide displays high variability in length and amino acid composition, and has been considered as an inert byproduct of insulin synthesis and processing for many years. Recent studies have suggested that C-peptide can act as a bioactive hormone, exerting various biological effects on the pathophysiology and treatment of diabetes. In this study, we analyzed the coevolution of insulin molecules among vertebrates, aiming at exploring the evolutionary characteristics of insulin molecule, especially the C-peptide. We also calculated the correlations of evolutionary rates between the insulin and the insulin receptor (IR) sequences as well as the domain-domain pairs of the ligand and receptor by the mirrortree method. The results revealed distinctive features of C-peptide in insulin intramolecular coevolution and correlated residue substitutions, which partly supported the idea that C-peptide can act as a bioactive hormone, with significant sequence features, as well as a linker assisting the formation of mature insulin during synthesis. Interestingly, the evolution of C-peptide exerted the highest correlation with that of the insulin receptor and its ligand binding domain (LBD), implying a potential relationship with the insulin signaling pathway.
PLoS ONE 01/2012; 7(12):e52847. · 4.09 Impact Factor
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ABSTRACT: In the present study, a total of 158 fecal samples were collected from diarrheal dogs younger than 1 year old in pet clinic in China. 20 specimens (20/158, 13%) were positive for Torque teno canis virus DNA using detection PCR. One representative positive isolate designated LDL was randomly selected, cloned and sequenced. The complete genome of the LDL Chinese strain was 2799 nucleotides in length and contains three open reading frames (ORFs), which encode 576 (ORF1), 101 (ORF2), and 243 (ORF3) aa. Compared with the human and other animal TTV genomes, the genome of the LDL strain is clearly smaller and shares 95% identity with Japanese cf-TTV10 strain (AB076002). Phylogenetic analysis showed that the present Chinese Torque teno canis virus LDL strain was also closely clustered with the previous Japanese cf-TTV10 strain, and formed a different branch together with Torque teno sus viruses 1 and 2 compared with other Torque teno viruses, Torque teno mini virus, and Torque teno midi virus. Our study demonstrated that Torque teno canis virus is present in China.
Virus Research 05/2011; 160(1-2):98-101. · 2.94 Impact Factor
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ABSTRACT: Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. TSOL18 has been identified as a host-protective oncosphere antigen. To obtain mouse monoclonal antibodies (mAbs) against TSOL18 and to map its antigenic epitopes are potentials to develop a vaccine for the prevention of T. solium infection. In this study, mAbs were produced by the hybridoma technique using purified glycosylated TSOL18 produced in Pichia pastoris as the immunogen. mAb was used to define the B-cell epitopes of TSOL18 with phage-displayed random dodecapeptide library (Ph.D.-12), and some of the positive phage clones were sequenced and analyzed. The predominant mimotopes were ETTKLQRFQAML (L1) found in 83%, followed by DHTXF in 15% (L2: DHTLFAASHNHR, DHTLFSTGHSHG, and DHTFMQRYHTHQ). Comparison of the peptide sequences with native TSOL18 protein sequence using Clustal W software showed that they did not completely match, suggesting that the ETTKLQRFQAML and DHTXF sequences should be conformational epitopes. The sera of mice immunized with the selected phage clones obviously recognized the TSOL18 protein. Meanwhile, sera collected from TSOL18-vaccinated pigs reacted to both epitopes in enzyme-linked-immunosorbent serologic assay test. Our work demonstrated that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAbs and a mimotope of TSOL18, which could provide an alternative approach for the diagnosis and development of a vaccine for T. solium.
Parasitology Research 02/2010; 106(5):1151-7. · 2.15 Impact Factor
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ABSTRACT: Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates.
Clinical and vaccine immunology: CVI 01/2009; 16(2):230-2. · 2.37 Impact Factor
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ABSTRACT: To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 09/2008; 24(8):1490-5.
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ABSTRACT: Taenia solium 45W proteins are good candidates for development of anti-cysticercosis vaccines. However, the genetic characteristics of the 45 gene family are still unclear between different isolates. We investigated the polymorphism of the 45 gene family between Chinese and Mexican T. solium. Alignment showed that TSO45-4B and TSO45-1C antigens were conserved absolutely, whereas other TSO45 proteins varied between these two isolates. It is informative to guide using of recombinant 45W vaccines to control porcine cysticercosis caused by Asiatic or African/Latin American T. solium.
The American journal of tropical medicine and hygiene 07/2008; 78(6):946-8. · 2.59 Impact Factor
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ABSTRACT: Cysticercosis caused by Taenia solium metacestodes is a worldwide public health problem. Important progress in the development of effective and practical vaccines against this disease has been made. In this study, the promising T. solium oncospheral vaccine candidate named TSOL18 antigen was produced in a 5-liter fermentor. During the process of fermentation, the pH of the culture was always kept below 5.0, and in order to prevent foaming, an antifoam agent was added. In addition, the oxygen content of the culture was constantly kept at >50% in our experiment. A high level of the glycosylated protein (2.5 g/liter) was obtained, and the protein was easily purified by gel chromatography. Vaccination trials showed that the recombinant TSOL18 antigen induced 94 and 100% reductions in metacestode burdens in vaccinated pigs, obviously higher than the 89% reduction in pigs immunized with cysticercus crude extracts in trial 1. These are very promising results in the development of an efficient tool to control cysticercosis in Asia.
Infection and immunity 03/2008; 76(2):767-70. · 4.21 Impact Factor
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ABSTRACT: An unknown gene, SLC10, was cloned by spliced leader-based polymerase chain reaction from Taenia solium. The full length of SLC10 was found to be 635 bp, encoding an 18.223 kDa protein. ELISA results showed that none of 70 normal and 75 cysticercosis sera samples reacted with purified recombinant SLC10 protein. Using an immunohistochemical method, it was revealed that the native SLC10 protein distributed extensively in inner cyst walls but not in the scolex in Cysticercus cellulosae. Together with predicted results, it is suggested that the SLC10 protein is a non-secretory structural protein, which is not involved in induction of the host's immune reactions against infection at least at the larval stage.
The Veterinary Journal 02/2008; 175(1):96-101. · 2.24 Impact Factor
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ABSTRACT: dUTPase plays an essential role in pyrimidine metabolism in many organisms. In this study we report the dUTPase-encoding gene (Tdut) from Taenia solium oncospheres and larvae. Alignment reveals that the putative protein contains five conserved motifs that are often found in many characterized dUTPases. The deduced amino acid sequence has only 65.2% identity with human dUTPases. This low identity encourages its use for the design of new drugs against cysticercosis.
Acta Tropica 04/2007; 101(3):266-70. · 2.72 Impact Factor
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ABSTRACT: The partial 18S rRNA sequences of E. coelmaticum and E. pancreaticum were amplified using conserved primers and an evolutionary tree was constructed using Neighbor-Joining. The percent identity of Eurytrema species with other Dicrocoeliidae varied from 97.5 to 98.2, while the percent identity between the two Eurytrema species was up to 99.3. The tree showed that E. coelmaticum and E. pancreaticum were not situated in the same position, and they formed one cluster with L. collurioni. These results support a confirmation with molecular data that E. coelomaticum and E. pancreaticum are different species which apparently were not seriously questioned in the past.
Parasitology Research 03/2007; 100(3):645-6. · 2.15 Impact Factor
