The Regulatory Effect Of Recombinant Aedes Albopictus Saliva RAlb-34k2 Protein On BHK Cells And Raw264.7 Cells Infected With DENV-2:A Preliminary Study

Objective:To investigate the possible regulatory effect of recombinant Aedes albopictus salivay 34k2 protein?rAlb-34k2?on DENV-2-infected BHK cells and Raw264.7 cells.Methods:1)The proliferation and cytopathic effect?CPE?of BHK and Raw264.7 cells,which were treated with rAlb-34k2 protein?10?g/ml,1?g/ml?and with/without DENV-2 under different MOI,were detected by RTCA technology.2)The expression difference of viral NS5 gene compared between rAlb-34k2?10?g/ml,1?g/ml?intervention group and virus control group,heat inactivated protein group and rabbit anti-ralb-34k2 antibody blocking group under the different MOI infection models,was detected by qRT-PCR method.3)The CIT₅₀-TCID₅₀0 standard equation was constructed by half cell index time(CIT₅₀)of BHK cells infected by DENV-2 in different TCID₅₀0 titer and the DENV-2 titer in the cell culture supernatant was eveluated by this equation.4)The effect of rAlb-34k2protein?1?g/ml?on mRNA expression difference of IFN-?/?,IL-1?,TNF-?,IL-10and CXCL2,CXCL10 in Raw264.7 cells within 18h,was detected by qRT-PCR method.Results:1)RTCA results showed that rAlb-34k2 protein?10?g/ml?1?g/ml?had no significant effect on the growth of BHK and Raw264.7 cells.2)When DENV-2 infects BHK cells?MOI=0.01,0.005?and Raw264.7 cells?MOI=5,10?,the different concentrations of rAlb-34k2 protein caused a leftward increase in the cell proliferation curve,wherein the protein intervention group?10?g/ml?induced the CIT₅₀0 of BHK cells shortened significantly?P<0.05?,the maximum CI value(CI_max)and the CI value at 45h(CI₄₅)of Raw264.7 cells shortened significantly?P<0.05?.3)Intracellular DENV-2 NS5 nucleic acid detection showed:After the intervention of rAlb-34k2 protein,the expression of NS5 nucleic acid in BHK cells and Raw264.7cells was higher than that in virus control group;In BHK cells,the high concentration protein group?10?g/ml?increased significantly.When MOI=1,it was 2.68 times?12h,P<0.01?,3.63 times?24h,P<0.01?and 1.62 times?48h,P<0.05?of the control group respectively;When MOI=0.5,it was 3.78 times of the control group?12h,P<0.01?;In Raw264.7 cells,the protein intervention group?10?g/ml?was 5.2 times?6h,P<0.01?and 3.5 times?12h,P<0.05?respectively in the viral infection group?MOI=5?;The inactivated rAlb-34k2 protein group and the specific antibody blocking group had not different from the virus control group.4)The RTCA standard equation was y=-7.007x+75.546,R²=0.9854;The comparison of the effectiveness and sensitivity of virus content?logTCID50/ml?showed:The live DENV-2 titers(logTCID₅₀/ml)were4.90±0.05?D2?,6.18±0.20?D3?and 6.04±0.10?D4?calculated by RTCA standard curve method,while its titers were 6.25±0.49?D3?and 5.87±0.35?D4?by traditional TCID₅₀0 method,respectively.It shows no statistically significant difference between the two methods?P>0.05?but RTCA standard curve method is more sensitive.The detection of live virus in the supernatant showed that the level of live virus in the supernatant of the protein intervention group was significantly lower than that of the virus control group at 48h.5)The rAlb-34k2 protein had no effect on the expression of IFN-?/?and TNF-?in Raw264.7 cells before and after infection;but it may up-regulate the expression of IL-10 in Raw264.7 cells and inhibit the expression of CXCL10 before infection;and inhibit the expression of IL-1?and CXCL2 in the course of infection.Conclusion:1)The rAlb-34k2 protein promotes the proliferation of DENV-2 in BHK cells and regulates the release of the virus.2)The rAlb-34k2protein can promote the proliferation of DENV-2 in Raw264.7 cells.3)The CIT₅₀-TCID₅₀0 standard curve method can be used to detect the viral titer of DENV-2with a higher sensitivity than the traditional TCID₅₀0 method.The experimental results provided a certain experimental basis for further investigation of the biological effects of rAlb-34k2 protein in DENV-2 infected target cells.