家蚕杆状病毒表面展示SARS-CoV的RBD蛋白及其免疫原性研究

利用杆状病毒表面展示技术,构建展示SARS-CoV的棘突蛋白（spike protein）受体结合结构域（receptor binding domain,RBD）的重组杆状病毒vBmgp64-RBD,并进一步研究其展示目的蛋白的免疫原性。结果表明：RBD蛋白正确表达并成功展示在病毒囊膜表面,将灭活的纯重组杆状病毒vBmgp64-RBD皮下注射BALB/c小鼠能产生抗RBD蛋白的特异性抗体,且抗体具有抗RBD蛋白的中和作用。证明vBmgp64-RBD有可能作为一种基于杆状病毒的新型SARS疫苗,为SARS的体外检测及后续疫苗的研究开发奠定了基础。     

Recombinant Baculovirus: A Flexible Drug Screening Platform for Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.     

利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究

研究了检测斑节对虾(Penaeus monodon)的主要致病病原——对虾白斑综合征病毒(white spot syndrome virus,WSSV)及斑节对虾杆状病毒(monodon baculovirus,MBV)的技术。用多重PCR检测方法,设计了两对特异性引物,从不同虾池中收集斑节对虾,提取DNA模板,同时检测两种对虾病毒。研究结果表明：该方法检测灵敏度高、特异性好,可检测至每毫克组织100个病毒粒子;从对虾组织中提取的DNA模板对病毒DNA的扩增无抑制,适合于对虾中两种病毒的同时检测。     

重组杆状病毒感染悬浮昆虫细胞感染策略优化及扩大试验

昆虫细胞-杆状病毒表达系统已经广泛应用于生物杀虫剂及重组蛋白的大规模生产。在昆虫细胞-杆状病毒表达系统中,感染策略中的感染复数(MOI)、感染时间(TOI)、细胞初始浓度(ICD)的相互关系还不是很清楚,而且相关报道也很少。本研究在摇瓶中利用正交实验探讨了MOI、TOI及ICD的相互作用关系以及对包涵体病毒(OV)和非包涵体病毒(BV)产量的影响。结果表明,感染复数对OV和BV产量有显著的影响;最佳感染条件为MOI为0.1,TOI为细胞处于对数生长期初期,ICD为2×105cells.ml-1;最大BV病毒滴度为2.0×107TCID50.ml-1。将在摇瓶中获得的最佳条件在气升式反应器进行扩大试验,获得最大病毒滴度为2.13×107TCID50.ml-1。     

杆状病毒在气升式反应器中感染策略优化

为了得到杆状病毒感染悬浮昆虫细胞最佳条件,利用正交实验探讨了气升式反应器中感染复数(MOI)、感染时间(TOI)、细胞初始浓度(ICD)及体系中的溶氧值(DO)的相互作用关系以及对包涵体病毒(OV)和非包涵体病毒(BV)产量的影响.结果表明:最佳感染条件为MOI为0.1,TOI为细胞处于对数生长期晚期,ICD为2×105cell·mL-1,DO为10%,最大BV病毒滴度为7.943×106TCID50·mL-1,最大OV病毒滴度为1.68×106OVs·mL-1.     

约氏疟原虫Pys25和Pys48抗原的表达与产物活性初步研究

通过家蚕杆状病毒表面展示技术获得约氏疟原虫有性阶段候选抗原Pys25（217AA）和Pys48（455AA）,以期建立一种新的鼠疟模型。首先利用大肠杆菌原核表达系统表达这两种抗原,免疫Balb/c小鼠制备多克隆抗体,同时利用家蚕杆状病毒表面展示技术,并改造pFastBac Dual载体,在其Ph启动子前端加入哺乳动物启动子CMV,并将目的蛋白基因与杆状病毒囊膜蛋白gp64基因片段融合表达,融合蛋白展示在病毒粒子表面。用其免疫Balb/c小鼠,在小鼠体内,CMV启动子启动融合蛋白表达,共同刺激小鼠产生多克隆抗体。通过间接ELISA检测,两种抗原抗体效价均能达到1∶12 800。本实验为后期免疫阻断效应研究、多时期多价疫苗的构建及鼠疫模型的建立提供了实验基础。     

Identification of Rhopalosiphum Padi Virus 5′ Untranslated Region Sequences Required for Cryptic Promoter Activity and Internal Ribosome Entry

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.