白纹伊蚊唾液ralb34k2-1对DENV2在不同细胞中复制的影响  

The change of DENV2 replication in different host cells treated with ralb34k2-1

作　　者：李莹 黄贞植 覃燕春 朱亚妮 吴家红 商正玲 LI Ying;HUANG Zhenzhi;QIN Yanchun;ZHU Yani;WU Jiahong;SHANG Zhengling(Department of Immunology,College of Basic Medicine,Guizhou Medical University,Guiyang 550025,China;Laboratory department,Joint Logistics Support Force No.940 Hospital of PLA;Department of Human Parasitology,College of Basic Medicine,Guizhou Medical University)

机构地区：[1]贵州医科大学基础医学院免疫学教研室,贵州贵阳550025 [2]联勤保障部队第九四○医院检验科 [3]贵州医科大学基础医学院寄生虫学教研室

出　　处：《中国病原生物学杂志》2023年第6期683-688,694,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81960295)。

摘　　要：目的探讨白纹伊蚊ralb34k2-1对DENV2在不同宿主细胞中复制的影响。方法采用Dot-ELISA和间接ELISA检测ralb34k2-1与DENV2/DENV-EDⅢ的结合特性。采用qRT-PCR检测不同剂量ralb34k2-1对蚊细胞C6/36、Aa23T,哺乳动物细胞BHK-21、RAW264.7、HacaT、THP-1中DENV2-E基因表达的影响;检测C6/36细胞中抗菌肽DEFE、DPT以及RAW264.7、THP-1细胞中抗病毒因子IFN-β、ISG15、ISG56基因表达的影响。结果Dot-ELISA和间接ELISA检测显示ralb34k2-1能与DENV2、DENV-EDⅢ结合,其中与DENV-EDⅢ结合的A值最高,为0.9177±0.04422。ralb34k2-1预孵育可促进DENV2在C6/36、Aa23T细胞中的复制(2^(-ΔΔCT)值最高为2.0419±0.1027)。5μg ralb34k2-1对C6/36中DEFE的抑制率可达73%(P<0.05)。ralb34k2-1能促进HacaT、THP-1细胞中DENV2的复制,瞬时混合组DENV-E2^(-ΔΔCT)值最高,为2.8024±0.3091,高于预孵育组的2.1325±0.06746,但对鼠源细胞BHK21、RAW264.7中DENV-E基因的表达无显著影响(均P>0.05)。抗病毒因子检测显示,ralb34k2-1单独作用能诱导RAW264.7、THP-1中IFN-β、ISG15、ISG56的表达,其中5μgralb34k2-1作用于RAW264.7细胞后ISG56RNA相对表达(2^(-ΔΔCT))值为377.9795±12.5457,THP-1细胞ISG56RNA的相对表达(2^(-ΔΔCT))值为9.5703±1.0332。结论ralb34k2-1可与DENV2、DENV-EDⅢ结合,推测该蛋白可能通过抑制蚊细胞中抗菌肽的表达协助蚊源细胞中DENV2的复制,而在人源细胞中ralb34k2-1协助DENV2复制的机制与蚊源细胞不同,可为伊蚊雌蚊唾液中34k2蛋白参与DENV2在蚊-人细胞中循环的研究提供实验线索。Objective To investigate the effect of ralb34k2-1 on DENV2 replication in different cells derived from mosquito and mammal.Methods 1)The binding characteristics of ralb34k2-1 with DENV2/rEDIII was evaluated by Dot-ELISA and indirect ELISA.2)DENV2-E gene expression in mosquito cells C6/36 and Aa23T and mammal cells BHK-21,RAW264.7,HacaT and THP-1 cells treated with ralb34k2-1 was detected by qRT-PCR,DEFE、DPT genes expression in C6/36 and IFN-β,ISG15,ISG56 treated with ralb34k2-1 in RAW264.7 and THP-1 was detected by qRT-PCR.Results 1)The binding results showed that ralb34k2-1 could interact with DENV2 and DENV-EDⅢ.The highest binding A value of ralb34k2-1 combined with DENV2 is 0.08775±0.01436,and the highest A value of Ralb34k2-1 combined with DENV-EDⅢwas 0.9177±0.04422.2)The mRNA expression of DENV-E in C6/36 and Aa23T cells pre-incubated with ralb34k2-1 were increased(2^(-ΔΔCT)value was 2.0419±0.1027).The inhibitory rate of 5μg ralb34k2-1 on DEFE in C6/36 was 73%(P<0.05).3)The mRNA expression of DENV-E of DENV2 was increased in HacaT and THP-1 cells infected with DENV2 mixed with ralb34k2-1(The 2^(-ΔΔCT)values was 2.8024±0.3091 which mixed instantaneously with ralb34k2-1 while pre-incubated with ralb34k2-1 was 2.1325±0.06746.But it had no significant effect on the expression of DENV-E gene in BHK-21 and RAW264.7 cells(P>0.05).4)The mRNA expression of IFN-β,ISG15 and ISG56 was induced by ralb34k2-1 strongly whether virus mixed in it or not.The mRNA relative expression of ISG56 in RAW264.7(377.9795±12.5456)was higher than that in THP-1(9.5703±1.0332).There was no significant difference in antiviral factors between ralb34k2-1 instantaneous mixing and pre-incubated THP-1(P>0.05).Conclusion The ralb34k2-1 can bind to DENV2 and DENV-EDⅢ,and may up-regulate the replication of DENV2 in mosquito cells by inhibiting antimicrobial peptides.Moreover,the replication of DENV2 in human cells enhanced by ralb34k2-1 is different from that in mosquito-derived cells.These results provides a novel paradigm fo

关 键 词：白纹伊蚊 重组34k2蛋白 DENV2 蚊源细胞 鼠源细胞系 人源细胞 

分 类 号：R384.1[医药卫生—医学寄生虫学]