The Effect Of Deletion Mutant △IPS-1 To IFN-β Induction And Its Antiviral To HSV-1

Objective:To construct the deletion mutant gene of IFN-βpromoter stimulator 1(△IPS-1) by depleting the amino acids region from the 300 to 444 loci and the recombinant plasmids pEF-BOS-FLAG/△I PS-1, study the effect of deletion mutant△I PS-1 to IFN-βinduction and its antiviral to HSV-1 initially by cell experiment in vitro and to further explore the contribution of the domain to IPS-1 executing the normal function and supply proof to research the function of IPS-1.Method:Primers were designed by Primer5.0 software, then overlap extension PCR were used to amplify the mutant IPS gene(△IPS-1) from pEF-BOS-FLAG/IPS-1 and the depleting mutant IPS-1 gene was cloned into pEF-BOS-FLAG. The constructed recombinant plasmids pEF-BOS-FLAG/△IPS-1 was identified by XbaⅠ/ClaⅠdigestion and DNA sequencing and were transfected to HEK293T by the calcium phosphate precipitation method. The expression products of the pEF-BOS-FLAG/IPS-1 and pEF-BOS-FLAG/△I PS-1 recombinant plasmids in HEK293T cell line were analyzed by western-blot analysis. ELISA was used to detect IFN-βsecretory volume in the supernatant of the cells culture medium after stimulating of IPS-1 and△IPS-1 at different time pionts. After transfected with different plasmids, HEK293T cells were infected by HSV-1 at different multiplicity of infection (MOI), the cytopathic effect (CPE) of the different plasmids transfected cell groups were determined by amido black stainning. The viral titer of the supernatant of the cells culture medium in different plasmids transfection groups at different time pionts were tested by plaque assay.Results:The depleting mutant IPS-1 gene and eukaryotic expression vector pEF-BOS-FLAG/△I PS-1 were constructed successfully. The result of enzymatic digestion identification and DNA sequencing indicated that the sequence of△IPS-1 gene was correct. Forty-eight hours after transfection of recombinant plasmids pEF-BOS-FLAG/△I PS-1 or pEF-BOS-FLAG/IPS-1 into HEK293T, western-blot analysis showed the positive lanes which relative molecular weight was about 44.2kDa and 58.8kDa and the protein molecular weight was consistent with△I PS-1 and IPS-1 respectively. Compared with the secretory volume of IFN-βin the supernatant of the cells culture medium transfected with the pEF-BOS-FLAG/△IPS-1, pEF-BOS-FLAG/IPS-1 and the pEF-BOS-FLAG , there were remarkable difference at different time pionts(P<0.001). The secretory volume of IFN-βin the pEF-BOS-FLAG/△I PS-1 group was lower than the pEF-BOS-FLAG/IPS-1 group at different time points and the statistical result had remarkable difference (P<0.05). Amido black stainning results showed the CPE in transfection group of pEF-BOS-FLAG/△I PS-1 was more than those in the pEF-BOS-FLAG/IPS-1 group. The plaque assay showed the viral titer in the pEF-BOS-FLAG-△IPS-1 transfection group was higher than the pEF-BOS-FLAG-IPS-1 transfection group, and the viral titer of the two groups were lower than the control group.Conclusion:(1)The deletion mutant gene△IPS-1 and the recombinant plasmids pEF-BOS-FLAG/△IPS-1 were constructed successfully.(2)△I PS-1 could decrease the secretory volume of IFN-βin HEK293T cells and could not completely suppress the CPE of the cells infected by HSV-1. The antiviral function of△IPS-1 to HSV-1 was weaken obviously.