Different Expression Gene Of CSFV Shimen Strain Cytopathogencity In Swine Veins Endothelial Cells And CSFV E2 PK-15 Cells Expression

Classical swine fever(CSF) is a kind of acute and intense contagious disease in swine, it material damages swine industry. CSF could cause swine appearing disseminated hemorrhage in systemic organ and tissue, degeneration and necrosis in small vascular and capillary endothelial cells. Classical swine fever virus (CSFV) belongs to genera Pestivirus, family Flaviviridae, it's genome total length is 12.3Kb,including a big ORF, encoding 11 structural and nonstructural proteins. CSFV has affinity to hematopoietic and blood vessel tissue. By pathological histology test, hemorrhage spots appeared in tissue and organ were more caused by occurrence of swelling, degeneration and necrosis in small vascular and capillary endothelial cells;with arteriole endothelial cells degenerating, necrosis and exfoliating, tunica intima became coarse, thrombus was formed, and stenosis or obliteration occurred which caused infarction nidus. Furthermore, myocardium appeared parenchyma degeneration, but parenchyma cells of kidney and other organs had no change. At present, most study showed CSFV was replicated and proliferated in vitro through swine kidney cells such as PK-15, PK-2a and SK-6 cell lines, and swine lung, spleen, testis and marrow other cells, but not caused the cytopathic effect (CPE) in general condition. By pathological histology observation, swine endothelial cells appeared obvious CPE after infected CSFV. Swine endothelial cell were cultured in vitro, shimen virulent strain of CSFV were inoculated endothelial cells, and observation of pathologic change of infected cells were done. Gp55 protein encoded by CSFV E2 gene is a major protective antigen of CSFV, and best material utilized to detect CSF and source of gene engineering vaccine presently. Based in affinities of genetic codes in gene expression, E2 gene was expressed in PK-15 cell line to increase productions of recombinant proteins expressed; again, post-processing in PK-15 cell from swine has obvious priority contrast to other host cells, the immunogenicity and immunity protective capacity of the productions would be increased, which offers basis for putting CSFV gene engineering vaccine into actual application. The study can be divided into three parts. first, to establish in vitro culture method of swine umbilicus veins endothelial cells, and study pathologic change on endothelial cell after infected CSFV shimen strain, and discuss molecular base of cytopathogencity of CSFV to swine umbilicus veins endothelial cells. Second, to analysis different gene expressed in up and down of CSFV cause CPE in swine endothelial cell, the results showed 11 sensitive genes may be responsible for cytopathogencity of CSFV to swine umbilicus veins endothelial cells. Finally, expressed CSFV E2 gene in PK-15 cell as receptor cell, which offers basis of establishment of CSFV test method using recombinant protein as antigen and utilization of CSF gene engineering vaccine. 1. swine umbilicus veins endothelial cells were isolated and cultured by callagenaseâ… as Jaffe's perfused method. The result showed the endothelial cells were observation by scanning electron microscope and were stained positively for factor â…§relative antigen. It shows cells cultured are endothelial cell. Endothelial cell were inoculated by CSFV shimen strain, we find endothelial cells appeared CPE at 36h after infected, and it became obvious at 48~72h. But PK-15 infected by CSFV shimen strain had no CPE, it shows CSFV just cause CPE in cells which have affinities to CSFV, not cause CPE in all somatic cell. Morphology and organelle of endothelial cells infected by CSFV shimen strain were observesed by transmission electron microscope. PK-15 and swine umbilicus veins endothelial cells infected by CSFV shimen strain was detected by direct immuno-fluorescent staining method and PCR, the results showed both of two were positive. It display CPE appeared in infected endothelial cell were caused by inocubating CSFV shimen strain. 2.The analysis to E2 and P80 gene expressed in up and down of swine endothelial cell inoculated by CSFV shimen strain was done, in order to eliminate the possibility that occurrence of CPE due to virus variation. RNA isolated from swine endothelial cell infected by CSFV were detected by Northern blot, it just appeared complete virus genome special banding, no defective interference particle (DI), so we eliminate the possibility that DI cause CPE in swine endothelial cell. 3.DDRT-PCR were done to swine endothelial cell noinoculated CSFV and swine endothelial cell inoculated CSFV shimen strain at 48h, 54h, 72h.After different display silver staining, it appeared 36 different expressed bandings, by PCR, gained 25 single bandings, then by Northern blot, it gained 11 positive different expressed fragment. 4. The E2 gene of CSFV Shimen strain was cloned into the eukaryotic expression vector PEGPF-C1. The positive plasmids was transducted by liposome into PK-15 cell lines. Positive cell clone were selected by G418 and immunofluorescence. E2 gene inducted into positive cell clone was approved by PCR and immunofluorescence. Immunochemistry and ELISA detection showed that the fusion protein is CSFV E2 gene code protein, which offers basis of establishment of CSFV test method using recombinant protein as antigen and utilization of CSF gene engineering vaccine.