| In this research porcine reproductive and respiratory syndrome virus (PRRSV) Sichuan isolates of PRRSV-SCl and PRRSV-SC2 were identified by the method of cell culture, electron microscopy and RT-PCR respectively. ORF5 genes of these two isolates were amplified, cloned and sequenced. Eukaryotic expression plasmid (nucleic acid vaccine) of PRRSV-SCl, PRRSV-SC2-ORF5 and IL-2 were constructed and expression, tissue distribution, security, immunogenicity of SC2-ORF5 and ajduvant effect of IL-2 were studied systematically. Results were as follows:1. Sequencing of ORF5 gene of porcine reproductive and respiratory syndrome virus Sichuan isolates: In this research ORF5 of PRRSV-SCl and PRRSV-SC2 were sequenced after amplified by RT-PCR and cloned into pGEM-T vector. ORF5 of PRRSV-SCl and PRRSV-SC2 showed 98-99% nucleotide identities and 99% amino acid identities with strain ATCC VR-2332, 57% nucleotide identities and 54% amino acid identities with strain LV. Computer analysis by software BioEdit and THMM-2.0 indicates that these two isolates have six potential hydrophobic regions and two potential transmembrane helices and one signal peptide respectively. Phylogenetic tree analysis by Tree View software showed that both PRRSV-SCl and PRRSV-SC2 are closely related to VR-2332 strain, but not closely related to LV strain.2. Construction of eukaryotic expression plasmid of PRRSV-ORF5 gene and its expression in Marc-145 cell line: ORF5 of PRRSV-SCl and PRRSV-SC2 were inserted into the eukaryotic expression plasmid pcDNA3.1 (+) to construct the recombinant plasmid pcDNA-PRRSV-SCl-ORF5 and pcDNA-PRRSV-SC2-ORF5, then pcDNA-PRRSV-SC2-ORF5 was expressed in the Marc-145 cell line by cytofectene?transfection reagent kit processing and electroporation. ORF5 expression was detected by means of immunofluoescence. An intense fluorescence could be observed 23h post transfection in the Marc-145 cells transfected by electroporation, which is 3h earlier thanthat processed by cytofectene?transfection reagent. The highest expression product was detected 56h post transfection in the cells transfected with cytofectene?transfection reagent and (or) electroporation. Results showed that pcDNA-PRRSV-SC2-ORF5 was delivered efficiently by the methods of cytofectene?transfection reagent and electroporation respectively into the cell and expressed.3. Studies on distribution and security of PRRSV-ORF5 gene eukaryotic expression plasmid in pigs and mice: Recombinant plasmids of pcDNA-PRRSV-SC2-ORF5 were transferred into the Balb/C mice and piglets by route of intramuscular injection. Then ORF5 gene was detected by PCR from blood, brain, heart, liver, lung, spleen, kidney and muscle of mice and piglets different time post immunization (PI). Results showed that recombinant plasmid pcDNA-PRRSV-SC2-ORF5 distributed in other tissues of mice 3h post inoculation. pcDNA-PRRSV-SC2-ORF5 was detected in the brain 3h-43d PI, in the liver, lung, spleen, kidney from 3h to 70d PI, in the blood and heart 3h to 84d PI, and was detected from the muscle till 115d PI. ORF5 gene was also detected from the same tissues of piglets. Results also confirmed that no pcDNA-PRRSV-SC2-ORF5 was integrated into the chromosome of these two kinds of animals.4. Constructionn of pig IL-2 gene eukaryotic expression plasmid and its application: Eucaryotic plasmid of pcDNA-IL-2 containing pig interleukin 2 gene was constructed and immune responses of pigs immunized with pcDNA-PRRSV-SC2-ORF5 or pcDNA-PRRSV-SC2-ORF5 and pcDNA-IL-2 were observed. Results showed that pcDNA-IL-2 could enhance both humoral and cellular immune responses induced by pcDNA-PRRSV-SC2-ORF5.5. Establishment of indirect ELISA for detection of antibodies against PRRSV: An indirect ELISA for detection of antibodies against porcine reproductive and respiratory syndrome virus was established employing PRRSV-SC2 isolate as coating antigen and some serum samples were assayed. Results showed that the method is characterized by its specificity, sensitivity and stability. |
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