ABSTRACT
A double-molecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection. Two single-stranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences. The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon. Under the optimal conditions of 10 mmol/L MgCl2 , 20 mmol/L Tris-HCl (pH=8. 0), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L. The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method.
ABSTRACT
LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Subject(s)
Animals , Female , Mice , Antibodies, Viral , Blood , Bacterial Toxins , Genetics , Allergy and Immunology , Metabolism , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Enterotoxins , Genetics , Allergy and Immunology , Metabolism , Escherichia coli , Genetics , Metabolism , Escherichia coli Proteins , Gene Fusion , Genetics , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , MetabolismABSTRACT
A total of 189 stool samples from swine with diarrhea, collected in various porcine farms in the central region of China were tested for porcine enteric caliciviruses (PEC) member porcine sapoviruses (SaV) by reverse transcription polymerase chain reaction (RT-PCR) amplification using primers designed to detect porcine SaV. Selected amplicons were sequenced to establish phylogenetic relationships with reference strains. Porcine SaV were detected in 12.70% (24/189) of the samples. Phylogenetic studies based on partial RNA polymerase gene sequences indicated that the field strains of viruses isolated in China were closely related (75.6 88.3% identity) to the porcine SaV Cowden reference strain. These results provide evidence that caliciviruses of the genus sapovirus circulate in piglets in China, but further studies are needed to clarify their importance as cause of diarrhea. This is the first report of PEC in China.
ABSTRACT
Objective:Study on characteristics of two synthesizd peptides based on CSFV E2 protein. Methods:B cell epitopes of CSFV E2 antigen were predicted using accessibility and flexibility schemes, associated with antigenicity , secondary structure and multiple sites prediction. Two antigen peptides (Pep1 and Pep2) have been designed and synthesized and their reactivety were detected with 8 McAbs and antiserum against mE2 protein, then the peptides were conjugated with BSA and immunized rabbits respectively. Results:Both Pep1 and Pep2 could react with antiserum and McAb A11, Pep2 could interact with McAbD5 and McAbD8. Only Pep1 BSA conjugate can stimulate high level and specific antibodies.Conclusion: The peptide1 has good antigenicity.