鲤Pdcd6ip蛋白真核表达载体的构建及抗病毒功能研究  

PDCD6IP INHIBITED THE REPLICATION OF SPRING VIRAEMIA OF CARP VIRUS

作　　者：邵玲 张明辉 彭军辉 SHAO Ling;ZHANG Ming-Hui;PENG Jun-Hui(Shanghai Fisheries Technical Extension Station,Shanghai Fisheries Research Institute,Shanghai 200433,China)

机构地区：[1]上海市水产研究所,上海市水产技术推广站,上海200433

出　　处：《水生生物学报》2022年第4期529-536,共8页Acta Hydrobiologica Sinica

基　　金：上海市科技兴农重点攻关项目(2021-02-08-00-12-F00755);上海市青年科技启明星计划(21QA1408300);上海市自然科学基金(21ZR1457800);国家自然科学基金(31602196)资助。

摘　　要：为进一步研究程序性细胞死亡6互作蛋白Pdcd6ip(Programmed cell death 6-interacting protein)分子功能,探讨其表达对鲤春病毒血症病毒(Spring viraemia of carp virus,SVCV)增殖的影响,研究通过逆转录-聚合酶链式反应扩增得到两端带有Nhe I和EcoR I酶切位点的pdcd6ip编码片段,并将其克隆至真核表达载体pCI-neo上,构建了真核表达质粒pCI-pdcd6ip。获得的过表达质粒转染至EPC细胞后进行RT-qPCR和western blot检测Pdcd6ip的表达情况,并利用CCK-8法检测其对细胞增殖的影响。细胞转染后24h进行SVCV感染,检测Pdcd6ip过表达对SVCV增殖的影响。结果显示,Pdcd6ip蛋白真核重组质粒能够在EPC细胞中大量表达,转染后72h细胞活性较对照组无显著差异。同时,免疫荧光、RT-qPCR和western blot检测结果均显示,Pdcd6ip蛋白过表达后,SVCV M蛋白mRNA水平和蛋白表达水平较对照组显著下降,表明Pdcd6ip过表达显著抑制了SVCV的增殖。研究为抗SVCV药物的研发提供了新的研究基础和设计思路。Spring viraemia of carp virus(SVCV)is an OIE listed highly pathogenic agent of spring viraemia of carp(SVC),which can cause a high morbidity and mortality in several economically important Cyprinidae fish species.SVC outbreaks occur mostly in the spring with water temperature below 17℃.The mortality of infected young carp can reach up to 90%.Currently,there are no licensed vaccines or therapeutics for SVCV.Thus,elucidation of the SVCV induced host factors against viral infection and its anti-viral mechanisms would be helpful for the development of effective interventions.We previously found that programmed cell death 6-interacting protein(Pdcd6ip)was significantly upregulated in the SVCV-infected EPC cells based on proteomic analyses.Pdcd6ip,also known as Alix or Aip1,has been identified to function in a number of pathways.It is a central component of the endosomal sorting complexes,which is responsible for sorting membrane proteins into vesicles that bud into the multivesicular bodies.Meanwhile,it is involved in apoptosis through caspase 9 activation following endoplasmic reticulum stress.Reports also indicate Pdcd6ip play a key role in the regulation of viral replication in many viruses,such as HIV,EIAV and VSV.To determine the role of Pdcd6ip in SVCV replication,the common carp(Cyprinus carpio)pdcd6ip gene was amplified by reverse transcript-PCR,and the coding region of pdcd6ip was cloned into the pRSET-A prokaryotic expression vector.Recombinant protein was induced,purified,and used to immunize rabbits.The specificity of Pdcd6ip antibody was verified by western blot.Next,the plasmid pCI-pdcd6ip for eukaryotic expression of Pdcd6ip,was constructed and transiently transfected into EPC cells.The expression of Pdcd6ip was verified by RT-qPCR and western blot.The viabilities of the transfected cells were examined at 24h,48h and 72h post transfection with a CCK-8 kit.The results showed that overexpression of Pdcd6ip did not affect the proliferation of EPC cells.Then,the Pdcd6ip overexpressed cells were infected w

关 键 词：鲤春病毒血症病毒 Pdcd6ip蛋白 过表达质粒 细胞活性 抗病毒 鲤 

分 类 号：S941.4[农业科学—水产养殖]