Expression And Application Study Of Porcine Enteric Calicivirus Recombinant Capsid Protein

1. Molecular epidemiology of PECA total of 189 stool samples from swine with diarrhea, collected in various porcine farms located in the central region of China were tested for porcine enteric caliciviruses (PEC) by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Selected amplicons were sequenced to establish phylogenetic relationships with reference strains. PEC were detected in 12.70% (24/189) of the samples. Phylogenetic studies based on partial RNA polymerase gene sequences indicated that the China isolates were most closely related (75.6%—88.3% identity) to the sapovirus (SaV) Cowden reference strain. These results provide evidence that caliciviruses of the genus SaV circulate frequently in piglets in China but further studies are needed to clarify their importance as cause of diarrhea. Whereas another PEC member porcine norovirus (NoV) was failed to be detected. This is the first report of PEC of China.2. Cloning and expression of porcine SaV capsid protein geneAccording to the capsid gene sequence of porcine SaV accessed on GenBank, a pair of primers was designed. Then capsid gene products were amplified from field strain isolated from piglet suffered from diarrhea. After purification, ligation and transformation, capsid gene was successfully cloned into pMD18-T, the cloned gene was confirmed to have 81.3%-91.3% identity in gene sequence level with other capsid gene in GenBank. Then the cloned capsid gene was subsequently sub-cloned into expressing vector pET28a(+) by using another set of primers via biological engineering methods and the resulting recombinant plasmid was then transformed into Rosetta(DE3) bacteria. It was confirmed by SDS-PAGE analysis and Western-blot that the recombinant Rosetta(DE3) bacteria induced by IPTG were able to express the capsid protein with molecule weight of 61kDa. The induced capsid protein account 20% of total bacterial protein.3. Development and application of indirect ELISA with recombinant capsid protein for detection of antibodies against porcine SaVAn indirect enzyme-linked immunosorbent assay (ELISA) was successfully developed to detect antibody to PEC based on a purified recombinant capsid protein of porcine SaV. The recombinant capsid protein antigen showed no cross-reaction with the positive sera of other porcine diseases. The ELISA was used to test against 42 sow serum samples and 96 young pig serum samples which collected from Hunan province. 83.33% (35/42) sow samples and 68.75% (66/96) young pig samples were tested positive. The indirect ELISA is suitable for large-scale epidemiological investigation for PEC infection.