Study On The Biological Function Of The Recombinant Ae.albopictus Saliva 34K2 Protein On Human Keratinocyte Infected With Dengue Virus Type 2 Strain(NGC)

Objective To purify recombinant34K2 protein(rAb-34K2)from Ae.albopictus(Guangzhou strain)salivary and to further investigate its possible biological functions on human keratinocytes(HacaT)infected with DENV2.Methods The prokaryotic rAb-34K2 Protein was expressed in pET-28-34K2 /E.coli BL21(DE3)by IPTG.Then analyzied the molecule weight,solubility,and specificity of rAb-34K2 protein by SDS-PAGE and Western blotting methods.Then,the rAb-34K2 protein was purified by Ni-affinity chromatography.The proliferation or cytopathic effect of rAb-34K2 protein on human keratinocytes(HacaT)infected with/without DENV2 were analyzed by RTCA.The RNA level of intracelluar DENVS partial NS1 gene and the mRNA level of IFN-?/?,IL-1?,TNF-? and IL-10 were detected by qRT-PCR during different infection stages and the apoptosis of HacaT treated with different concentration of rAb-34K2 or BSA with/without DENV2 infection was analyed by FACS.Results The rAb-34K2 protein was specifically expressed as inclusion form.After denatured and renatured by urea,the rAb-34K2 protein was purified and and its concentration was about 350?g/mL.The cell growth of HacaT treated without/with rAb-34K2 protein at the concentration of 1 ?g/mL,5 ?g/mL,10 ?g/mL has no significant difference.The CPE of infected HacaT has more severe because the cell index(CI)value was decreased significantly and CIT50 was shorter significantly at rAb-34K2 protein concentration of 10?g/mL(P<0.05).The intracelluar NS1/GAPDH RNA ratio was significantly higher in rAb-34K2(1or10?g/mL)treated group than in DENV2 control group after infected with MOI of 1 at 36 h and also significantly higher in rAb-34K2 treated group after infected with MOI of 0.1 at 48 h,60,70 h.The IFN-?/GAPDH mRNA ratio and IFN-?/GAPDH mRNA ratio was dramatically decreased in rAb-34K2 treatment group(P<0.05 and P<0.01 relatively),coupling with IL-1?/GAPDH mRNA ratio significantly up and TNF-?/GAPDH mRNA ratio markedly down after infected with MOI of 1 at 24 h.The apoptosis of HacaT treated with rAb-34K2 protein(1 or 10?g/mL)under infected with MOI of 1 was significantly more than that of DENV2 control group(P<0.05).However,the apoptosis of hacaT without DENV2 was not significantly changed after treated with 1?g/mL or 10?g/mL of r Ab-34K2 protein.Conclusion The rAb-34K2 protein derived from pET-28-34K2 recombinant plasmid was successfully induced and purified.The rAb-34K2 has no obvious effects on the growth of HacaT but can promote the proliferation of DENV2(MOI =1/0.1)in infected HacaT cells,regulate the expression of IFN-?/?,IL-1?,TNF-? and IL-10,induce HacaT apoptosis infected with DENV2.