The Regulation Of Chaperone Jiv90 On Classical Swine Fever Virus Replication

Molecular chaperones are highly conservative protein family in evolution, which play important roles in some functions of proteins, such as assisting the cells under normal and stressful circumstances as so to maintain cell homeostasis and participating in many physiological effect.The reason of using molecular chaperones in viruses, might be keeping their genomes small enough under the selective pressure.In order to accomplish the related processes of virus multiplication, a lot of viruses parasitized in eukaryotic and prokaryotic cells could directly use molecular chaperones in their host cells, or code the virus protein chaperones or some functional proteins. It is reported that, the molecular chaperone Jiv90 plays an important role in classical swine fever virus (CSFV) infection, which might be involved in the adjustment by the Jiv90 cleavage of CSFV NS2-3 protein.To further study the effect of the molecular chaperone Jiv90 in the infection process of CSFV, we constructed the eukaryotic expression vector pEGFP-C1-Jiv90 and siRNA vector expressing short hairpin RNA (shRNA)sections which inhibit the expression of Jiv90 gene. Results from this study formed some important basis for Jiv90 gene function research and provided some new ideas for future researches on CSF prevention. And the results as follows:(1)Primers were designed according to Jiv90 gene and pEGFP-C1 sequences, DNA fragments after RT-PCR recovered from agrose gel were inserted into pEGFP-C1 plasmid, successfully constructed expression vector pEGFP-C1-Jiv90. Then the recombinant plasmid was identified by restriction enzyme analysis and DNA sequencing. After the sequence alignment, it is showed that both the swine Jiv90 gene and the bovine Jiv90 gene had 95% homology at nucleic acid level, which indicated that the molecular chaperone had high homology between the two species and similar functions.(2)Those plasmids were amplified by transfecting the competent cells, and the concentration and purity of its solution were detected by spectrophotometer. Then the plasmid pEGFP-C1-Jiv90 was transfected into SUVEC by liposome, furthermore, the efficiency of transfection was identified by observing from the inverted fluorescence microscope, and the G418-resistant colonies were isolated and multiplied.After transfecting of pEGFP-C1-Jiv90, the mRNA expression of Jiv90 gene in SUVEC was detected by Real-time PCR, which was showed that, compared to the control groups, the experimental groups increased about 16.35 times, as means the gene expression were significantly enhanced. It is showed that cells infected with CSFV Shimen strain after 72 h, which already expressed Jiv90 gene in SUVEC, had an evidently higher mortality rate than control group.The results of Real-time PCR showed that CSFV RNA in SUVEC-Jiv90 compared to the control groups increased about 4.26 times when cells infected with CSFV shimen strain after 60 h.(3)Through software, RNAi sequences were designed by using 4 different sites of Jiv90 gene, and according to the designed sequences, sense and anti-sence DNA oligo were chemically synthesized. In the end, the plasmid vectors pGPU6/GFP/Neo-Jiv90 shRNA1, shRNA2, shRNA3, shRNA4 were successfully constructed by annealing and cloning into pGPU6/GFP/Neo.Then the recombinant plasmids were identified by restriction enzyme analysis and DNA sequencing. Also, Interfering plasmid from GAPDH was established as positive control and interfering plasmid targeting none genes served as negative control. Then the recombinant interfering plasmids were transfected into SUVEC by liposome and the G418-resistant colonies were isolated.