白纹伊蚊rAlb-34k2蛋白对C6/36细胞内的DENV2早期抑制作用

The Aedes albopictus salivary protein rAlb-34k2 inhibits DENV2 proliferation in C6/36 cells in the early stages

目的探讨白纹伊蚊唾液rAlb-34k2蛋白在DENV2感染C6/36细胞中的作用。方法采用RT-qPCR检测不同浓度rAlb-34k2蛋白作用6 h、60 h后C6/36细胞内DENV2 NS1核酸的变化;采用Dot-ELISA检测rAlb-34k2蛋白与DENV2颗粒结合情况;采用ELISA及Western blot分别检测rAlb-34k2蛋白与C6/36细胞膜蛋白结合的差异。结果rAlb-34k2蛋白作用6 h后,C6/36细胞内DENV2 NS1 RNA的相对表达被抑制,其中10、25、100μg/ml浓度组NS1 RNA的相对表达(2^(-ΔΔCT))值分别为(0.6778±0.04827)、(0.5972±0.06369)和(0.4704±0.05297),差异有统计学意义(P<0.05);rAlb-34k2蛋白作用60 h后,各蛋白组间差异无统计学意义。Dot-ELISA检测rAlb-34k2蛋白(71、118.3、177.5μg/ml)可与DENV2病毒颗粒结合。间接ELISA检测rAlb-34k2蛋白与C6/36细胞疏水性膜蛋白和亲水性膜蛋白结合的A值分别为(1.1±0.26)和(3.1±0.047),差异有统计学意义(P=0.0016),Western blot检测亲水性膜蛋白的阳性结合条带数显著多于疏水性膜蛋白。结论白纹伊蚊雌蚊唾液特异性rAlb-34k2蛋白体外能与DENV2病毒颗粒及C6/36细胞膜蛋白结合,并抑制DENV2病毒早期在C6/36细胞中增殖。

Objective To investigate the role of the Aedes albopictus salivary protein rAlb-34 k2 in C6/36 cells infected with DENV2.Methods 1)C6/36 cells were infected with DENV2 and treated with different concentrations of rAlb-34 k2 for 6 or 60 hours.The level of DENV2 NS1 mRNA in C6/36 cells was detected using RT-qPCR.2)The binding of the rAlb-34 k2 protein to DENV2 was measured using Dot-ELISA.3)The affinity of the rAlb-34 k2 protein for membrane proteins of C6/36 cells was determined using indirect ELISA and Western blotting.Results 1)The expression of DENV2 NS1 in C6/36 cells was inhibited by treatment with rAlb-34 k2 after 6 h.The 2^(-ΔΔCT) values for groups treated with the rAlb-34 k2 protein(10,25,or 100μg/ml)were 0.6778±0.04827,0.5972±0.06369,and 0.4704±0.05297,respectively(P<0.05).In contrast,there were no significant differences among the groups after 60 h.2)Dot-ELISA analysis indicated that the rAlb-34 k2 protein(71,118.3,and 177.5μg/ml)bound to DENV2 virus particles.3)Indirect ELISA assay indicated that the A value for rAlb-34 k2 protein bound to the hydrophilic membrane proteins of C6/36 cells(3.1±0.047)was higher than that for hydrophobic membrane proteins(1.1±0.26)(P=0.0016).Western blotting yielded many more bands indicating binding to hydrophilic membrane proteins than to hydrophobic membrane proteins.Conclusion The Ae.albopictus salivary specific protein rAlb-34 k2 bound to both DENV2 virus particles and C6/36 cell membrane proteins in vitro,and rAlb-34 k2 inhibits DENV2 virus proliferation in C6/36 cells in the early stages.