The Pilot Research To Correlation Of Jiv Protein Expression And Classical Swine Fever Virus Relication

The Jiv protein is a kind of intracellular molecular chaperones discovered in recent yearsand it was proved to be closely related to the infection of pestivirus. It is has been shown thatthe amount of Jiv protein directly affect the cutting efficiency of the plague virus NS2-3dimerprotein, thereby the effects of classical swine fever virus(CSFV) replication and infection byregulating the release of NS3protein monomer indirectly. To further investigate therelationship of Jiv protein expression and replication of CSFV, and preliminarily study therole of Jiv protein in CSFV infection, this investigation detecteded the expression of Jiv genein CSFV uninfected and infected swine tissues by developing a real-time RT-PCR fordetecting the expression of Jiv gene. Further more, it has detectected the RNA relication levelof CSFV in infected swine tissues and preliminarily research the correlation between the Jivgene expression and tissue tropism of CSFV. Besides, it investigated the correlation of Jivprotein expression and replication of CSFV by detecting the RNA replication level of CSFVin Jiv over-expressed and blank cell lines. This investigation obtains the following findings:1. The present study has successfully established a real-time RT-PCR based on SYBRGreenⅠfor detecting the expression of Jiv gene, and shows that the PCR has a good linearrelationship （r2=0.999）between10～107copys and high amplification efficiency (103.4%)from the standard curve. In addtion, the sensitivity and reproducibility of the method arefavorable by detecting the minimum concentration of Jiv gene (10copies) and calculating thevariation coefficient of intro-group and inter-group (both below0.5%).2. The Jiv expression of swine tissues has been increased after infecting with CSFV byapplying the mehod to detect the Jiv gene expression in uninfected and infected tissues.Further more, the expression of Jiv gene in swine spleen and lymph node has a higher level indifferent swine tissues. The study also has dectected the RNA replication levels of CSFV ininfected tissues and the results showed that the level in spleen and lymph node are also higher.Besides, the results of dection to Jiv gene in three virous of cell lines infected with CSFV indifferent period show that the Jiv gene expression trend is consistent with the level of viralreplication and the Jiv gene expression of ST cell lines in different infection period is higher than two othe kinds of cell lines.The above results preliminarily indicate CSFV infectionpossbily stimulates the expression of Jiv gene, and the tissue tropism of CSFV to spleen andlymph node possibly relates to the expression level of Jiv gene.3. The Jiv gene eukaryotic expression vector was successfully constructed, andtransfected to SUVEC cell lines, then screened positive cells by G418, the results ofquantitative PCR and Western blot showed that Jiv protein in SUVEC cell lines was overexpressed successfully. By infecting CSFV on cell lines and the results of quantitative PCRshowed that Jiv over expessed-cell lines (infected with CSFV after24h,48h,72h,96h) hadhigher level of CSFV replication than the control cells (non-transfected cells and cellstransfected with pEGFP-N1), in addtion, this divesity was more obvious at24h and48hafter infecting with CSFV on these cell lines, therefore it indicated that the regulation of Jivprotein to viral replication possibly occured in the early stages of infection.