Effect Of Cyclophilin A On Replication Of Japanese Encephalitis Virus

Cyclophilin A (CypA), the prototypical member of the cyclophilins family, is an 20 kDa partner protein.As first discovered cyclophilin, CypA can inter mediate cell signaling, involve in protein folding and the inflammatory response and so on. With the studying of CypA found that the expression of CypA related with a number of diseases, recent studies on the role of CypA related to viral replication is also increasing. This experiment focuses on the cyclophilin A effect on Japanese encephalitis virus (JEV) replication.The following tests were processed to study the influence of porcine CypA on JEV replication. 1. Construct the eukaryotic expression plasmid of porcine CypA gene and build the real-time fluorescence quantitative detection method of JEVAccording to gene sequences in GenBank of porcine CypA designed a pair of specific primers and upstream and downstream primers were added KpnI, XhoI restriction sites. Porcine CypA gene was cloned into pMD19-T simple vector.KpnI and Xhol restriction enzyme digested pMD19-T-CypA vector, recycling CypA gene and connecting to the pcDNA3.1 (+) vector which were digestion by Kpnl and XhoI to construct pcDNA3.1-CypA. Transformed DH5a competent cells, positive plasmid send to sequence analyse. Sequence analysis showed that porcine CypA gene insertion sites and direction entirely correct. According to gene sequences in GenBank of JEV designed a pair of specific primers to amplify the E gene of JEV, then the E gene gene was cloned into pMD19-T simple vector,as a standard sample to construct JEV fluorescence quantitative standard curve and did specificity, sensitivity and reproducibility of the test. The results showed that its specificity, sensitivity and reproducibility are better,and only JEV positive samples amplified "S" shaped curve,and detecting the minimum concentration of JEV were 6copies/μL.2. The effect of recombinant pcDNA3.1-CypA on JEV replication in VitroExtracting the recombinant plasmid pcDNA3.1-CypA, liposome transfected BHK-21 cells, the establishment of pcDNA3.1 (+) empty vector transfected group and is blank group. Using Westen blotting and immunofluorescence method to identify the expression of eukaryotic expression vector in cells.The two group that each was transfected with pcDNA3.1-CypA and pcDNA3.1 (+) and the blank group were infected with JEV-SC-1, then the infected cells and supernatant were collected in different times postinfection. JEV fluorescence quantitative RT-PCR was used to detect the vRNA contents of different samples, and the JEV one-step growth curve of different groups were drew respectively.The intracellular JEV one-step growth curves were similar in different groups,but extracellular one-step step growth curves had a big difference.0-20hpi. the JEV vRNA content had no significant differences between pcDNA3.1-CypA group and control groups; 20-72hpi, the JEV vRNA content of control groups were significantly lower than pcDNA3.1-CypA group; after infectioned pcDNA3.1 (+) group had no significant difference with the control group.3. The Influence of Porcine Cyclophilin A on JEV Replication in VitroThe BHK-21 cell was cultured with different culture medium which contain different concentration of porcine CypA after infected with JEV-SC-1, the infected cells and supernatant were collected in different times postinfection. JEV fluorescence quantitative RT-PCR was used to detect the vRNA contents of different samples, and the JEV one-step growth curve of different groups were drew respectively. The intracellular JEV one-step growth curves were similar in different groups,but the extracellular JEV one-step growth curves were a big difference in different groups. At 0-12hpi,there were no significant difference in the groups, at 12-72hpi, the three groups that added CypA protein the viral RNA levels were higher than the control group.In logarithmic growth phase, adding porcine CypA protein JEV proliferation faster than the control group. At 16hpi,0.3mg/mL and 0.5mg/mL two treatment groups viral RNA levels were higher than the control group, at 60-84hpi, O.lmg/mL group and the control group viral RNA levels had no significant difference. After infection 20-60h except the 36h,0.5mg/mLgroup viral RNA levels were higher than the O.lmg/mL group and control group.