机构地区:[1]中国海洋大学海洋生命学院,海洋生物遗传与育种教育部重点实验室,山东青岛266003 [2]中国海洋大学海洋生物进化与多样性研究所,山东青岛266003
出 处:《中国海洋大学学报(自然科学版)》2023年第11期65-78,共14页Periodical of Ocean University of China
基 金:国家重点研究发展计划项目(2018YFD0901301);山东省自然科学基金项目(ZR2020MC189)资助。
摘 要:为探究对虾白斑综合征病毒(WSSV)3种囊膜蛋白VP19、VP24和VP28对其假型昆虫杆状病毒的包装与对虾组织亲嗜性的影响,本研究首先分析了过表达的上述3种囊膜蛋白在包装细胞(Sf9)中的亚细胞定位,然后通过共转染的方法分别构建了其假型杆状病毒:Bacmid-GUS/VP19、Bacmid-GUS/VP24和Bacmid-GUS/VP28,比较了上述3种假型杆状病毒的包装效率及其在感染对虾组织上的特异性。结果表明,过表达的VP19、VP24和VP28均可定位于包装细胞的细胞膜上,均能促进假型病毒的包装效率,并分别提高了14.1%、11.5%和11.7%,且携带不同囊膜蛋白的假型病毒在感染对虾成体时具有不同的最低感染剂量和不同的组织特异性。其中,假型病毒Bacmid-GUS/VP19和Bacmid-GUS/VP24仅能感染对虾成体的心脏和肌肉组织,且在单个对虾肌肉组织中两者的最低感染剂量分别为1×1011、1×108个具有生物活性的病毒颗粒数。而假型病毒Bacmid-GUS/VP28不仅能感染对虾的心脏和肌肉组织,还能感染对虾的鳃组织,且其在单个对虾鳃组织中的最低感染剂量为1×108个转导单位,在单个对虾肌肉组织中的最低感染剂量为1×109个转导单位。病毒感染对虾的成体组织的电镜分析结果提示,同哺乳动物细胞中的感染结果相似,假型昆虫杆状病毒在对虾成体组织中的感染也是复制缺陷的。本研究成果拓展了人们对于对虾WSSV病毒囊膜蛋白的生物学功能的认知,为假型昆虫杆状病毒介导的基因转移与表达系统的构建及其在对虾活体基因转移中的应用提供指导。In order to explore the effects of three envelope proteins of VP19,VP24 and VP28 from shrimp white dot syndrome virus(WSSV)on the packaging and shrimp tissue-tropism of their corresponding pseudotyped insect baculoviruses,the subcellular localization of the over-expressed three envelope proteins mentioned-above in the packaging cells(Sf9)was first analyzed in this study,and then the corresponding pseudotyped baculoviruses of the above-mentioned three envelope proteins,which were Bacmid-GUS/VP19,Bacmid-GUS/VP24 and Bacmid-GUS/VP28,were produced by co-transfection,respectively,and the packaging efficiency and their tissue specificity in shrimps were compared.The results obtained showed that most of the over-expressed proteins of VP19,VP24 and VP28 had been localized in the membrane of the packaging cells,and in the same time the packaging efficiencies of these pseudotyped baculoviruses had increased by 14.1%,11.5%and 11.7%,respectively in comparison with the intact baculovirus(Bacmid-GUS).Moreover,these pseudotyped baculoviruses carrying various envelope proteins had different lowest infectious doses and different tissue-specificities when infecting adult shrimps.Among them,the pseudotyped viruses of Bacmid-GUS/VP19 and Bacmid-GUS/VP24 could only infect the heart and muscle tissues,and the lowest infectious dose for muscles was 1×1011 TU/ind.for Bacmid-GUS/VP19,and 1×108 TU/ind.for Bacmid-GUS/VP24.The pseudotyped virus of Bacmid-GUS/VP28 could infect not only the heart and muscle tissues,but also the gill tissue of shrimp with a lowest-infectious-dose of 1×108 and 1×109 TU/ind.for muscle tissues,respectively.The results by transmission electron microscope suggested that,similar to the infection of baculovirus in mammalian cells,the infection of the pseudotyped baculovirus mentioned above in the tissues of adult shrimps was also replication-deficient.The results obtained in the present study will expand our knowledge of the biological functions of shrimp WSSV envelope proteins,and give us a good guidance on the
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