ABSTRACT
Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs serve as the amplifying hosts. In the present study, the full genomic sequences of two JEV strains (HEN0701 and SH0601) isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains had an 88.8% nucleotide sequence similarity and 97.9% deduced amino acid sequence homology. HEN0701 had high nucleotide sequence and high amino acid sequence identity with genotype I (GI) strains, while SH0601 had high nucleotide sequence and high amino acid sequence identity with GIII strains at both the gene and full genome levels. Further phylogenetic analysis showed that HEN0701 belonged to the JEV GI group and SH0601 was classified as a GIII strain. Analysis of codon usage showed there were a few differences between the GI and GIII strains in nucleotide composition and codon usage for the open reading frames.
Subject(s)
Animals , Cricetinae , Cell Line , Encephalitis Virus, Japanese/classification , Encephalitis, Japanese/epidemiology , Gene Expression Regulation, Viral/physiology , Genome, Viral , Molecular Epidemiology , Phylogeny , Swine , Swine Diseases/epidemiologyABSTRACT
Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs serve as the amplifying hosts. In the present study, the full genomic sequences of two JEV strains (HEN0701 and SH0601) isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains had an 88.8% nucleotide sequence similarity and 97.9% deduced amino acid sequence homology. HEN0701 had high nucleotide sequence and high amino acid sequence identity with genotype I (GI) strains, while SH0601 had high nucleotide sequence and high amino acid sequence identity with GIII strains at both the gene and full genome levels. Further phylogenetic analysis showed that HEN0701 belonged to the JEV GI group and SH0601 was classified as a GIII strain. Analysis of codon usage showed there were a few differences between the GI and GIII strains in nucleotide composition and codon usage for the open reading frames.
Subject(s)
Animals , Cricetinae , Cell Line , Encephalitis Virus, Japanese/classification , Encephalitis, Japanese/epidemiology , Gene Expression Regulation, Viral/physiology , Genome, Viral , Molecular Epidemiology , Phylogeny , Swine , Swine Diseases/epidemiologyABSTRACT
ObjectiveTo immunoscreen the mimic peptides of Mycobacterium tuberculosis antigen from phage displayed 12-mer peptide library.MethodsSpecific IgG was purified from sera of patients with TB and used as the target to immunoscreen a phage random peptide library of 12 amino acids.Positive clones which were obtained after three rounds of biopanning were detected by ELISA and sequenced.The diagnostic value of the high frequent positive clones were observed by ELISA.Results After 3 rounds of immunoscreening,the eluted phages were enriched effectively.Six kinds of animo acid sequence were obtained from twelve positive phage clones.Sensitivity of the two high frequent positive clones were 71.4％ (A2)and 55.4％ (A7) respectively.ConclusionThe antigen-mimic peptide was successfully screened from 12 random phage peptide library and the peptides can be recognized by tuberculosis patients' polyclonal antibodies.
ABSTRACT
In recent years, mass outbreaks of highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) have spread all over the Chinese swine industry. Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV, pAPRRS, constructed in our group, we constructed several chimeric clones with various substitutions of structural protein genes (ORF4-7) and 3' UTR between attenuated pAPRRS and virulent pJX143.Upon transfection of MA-104 cultured cells, all chimeric constructs pSX12, p5NX12, and p56N12 were rescued. The rescued viruses maintained the similar virological properties, based on the results of the growth curve of the rescued viruses. To test if the chimeric viruses can be used as a vaccine candidate, vSX12 and v56N12 vaccinated pigs were challenged with the HP PRRSV JX143 strain. As a result, the vSX12 vaccinated pigs were all seroconverted by 14-day-post vaccination, while v56N12 vaccinated pigs showed poor antibody response. Upon challenge, the vSX12-vaccinated group showed no signs of clinical PRRS syndrome, and virema period was shorten to 6 days post-challenge. Our results demonstrated that 1) vSX12 chimeric virus is a good vaccine candidate; 2) the virulence determinants of HP PRRSV probably located in coding regions other than ORF3-7 and 3' UTR, as our chimeric viruses were proved to be attenuated.
Subject(s)
Animals , Cloning, Molecular , Open Reading Frames , Porcine Reproductive and Respiratory Syndrome , Virology , Porcine respiratory and reproductive syndrome virus , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Recombination, Genetic , Genetics , Swine , Vaccines, Attenuated , Allergy and Immunology , Viral Envelope Proteins , Viral Proteins , Genetics , Viral Vaccines , Allergy and ImmunologyABSTRACT
The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.
ABSTRACT
The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirns spike protein induced hormonal and cellular immune response in Balb/c mice.
ABSTRACT
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Plasmids/biosynthesis , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/genetics , Viral Vaccines/biosynthesisABSTRACT
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Subject(s)
Humans , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Membrane Proteins , Genetics , Plasmids , Genetics , Recombinant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus , Chemistry , Genetics , Viral VaccinesABSTRACT
Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.
ABSTRACT
Objective To analyze the influence of Eomecon chionanthe alkaloids(ECA)on differential expression of liver proteins from Oncomlania hupensis.Methods O.hupensis was immersed in 10 mg/L ECA or clean water for 36 h and livers were isolated from live snails.Total liver proteins were extracted and separated by two-dimensional gel electrophoresis,differentially expressed proteins between ECA group and clean water group were selected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and tandem mass spectrometry sequencing of tryptic peptides.Results There were(354?110)and(311?12)spots observed in ECA group and in water group,respectively.Eleven differential expressed proteins were identified and all were up-regulated in ECA group.The eleven identified proteins were hypothetical protein,ENSANGP00000022175,class I aldehyde dehydrogenase,unknown protein,ENSANGP00000027067,protein similar to ankyrin 3 isoform 1 isoform 4,protein similar to hepatitis A virus cellular receptor 1 short form,mitochondrial isocitrate dehydrogenase 2-like,protein similar to keratin,keratin-10,and keratin-1,respectively.Conclusion Expression of liver proteins from O.hupensis could be changed when immerse in ECA.
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0. 05) and the specificity is higher than that of the SEA-ELISA (P