新城疫病毒样颗粒的研究
摘要
本论文针对新城疫病毒(NDV)出芽机制不清楚和亚单位疫苗免疫效果不理想这一重要理论和实践问题，拟以病毒样颗粒(VLPs)技术为突破口，建立阐明NDV出芽的主要机制、全面评估新城疫病毒样颗粒(ND-VLPs)的免疫原性和免疫效力的技术平台，为开发新型、高效和安全的NDV疫苗和阐明新城疫病毒致病机理奠定基础。
本研究首先构建了可能与ND-VLPs形成有关的NDV国内标准强毒F48E9株的结构基因M、F和HN的哺乳动物细胞重组表达载体pGFP-M、pGFP-HN和pGFP-F，经过限制性内切酶分析、质粒PCR鉴定和序列分析，证明我们获得了相应基因的哺乳动物细胞重组表达载体阳性质粒。然后将pGFP~--M、pGFP~--HN、pGFP~--F和pGFP~--NP共转染仓鼠肾细胞(BHK21)，72小时后收集共转染的细胞和上清应用电镜检测到了与NDV类似的颗粒，数量很少，结构也不典型，可能是共转染和表达效率较低的原因。为了解决这一问题，我们又将这些质粒分别转染BHK21，经过选择性培养基筛选后，获得了分别含有pGFP~--M、pGFP~--HN、pGFP~--NP和pGFP~--F重组表达质粒的细胞系，克隆筛选工作尚在进行中。对第一代转染的细胞培养上清和细胞应用SDS-PAGE、RT-PCR和间接免疫荧光检测，均未获得阳性结果。
同时，为了研究ND-VLPs在昆虫细胞中以及细胞外形成的可能性，我们将M、F和HN基因亚克隆到pFASTBAC1载体上，从而成功地构建了重组杆状病毒转座载体pFASTBAC1-M、pFASTBAC1-F和pFASTBAC1-HN，并分别转化到DH10BAC感受态细胞中，通过筛选获得了重组的杆状病毒穿梭载体Bacmid-M、Bacmid-F利Bacmid-HN。将重组杆状病毒穿梭载体分别转染Sf9细胞，经PCR检测获得了重组的杆状病毒。重组的杆状病毒分别感染Sf9细胞，SDS-PAGE检测到了M和F蛋白的表达，但没有检测到HN蛋白的表达。
本试验中我们构建了两种表达系统来研究ND-VLPs的形成和组装，不仅为揭示NDV的出芽机制、各基因及基因内不同功能区在病毒山芽、复制与组装过程中的作用，而且为进一步研究NDV的分子生物学特性，研制高效力的多价亚单位疫苗奠定了基础。
As a efficient investigation method, Virus-Like Particles (VLPs) technique has play an important role in the study of character, polymorphism, vaccine of many viruses. So the application of VLPs technique will help to discover the budding mechanism, develop novel, efficient and safe vaccine of NDV.
We constructed the mammalian cells expressing vector pGFP- -M, pGFP- -HN and pGFP- -F based on the structure gene of F48E9 strain of NDV,which is correlative with the formation of ND-VLPs.Then the recombinant plasmid pGFP- -M, pGFP- -HN, pGFP--F and pGFP--NP were co- transfected into BHK21 cells , some particles like NDV can be observed by electronic microscopy in collected 72h-post co-transfected cells and culture media,but we couldn't draw a conclusion that it's just ND-VLPs. To increase the efficiency of co-transfection, pGFP--IVK pGFP- -HN and pGFP'-F were transfected into BHK21 cells respectively, and screening with G-418 selective media, the result show that we have ot the stable cell line harbouring the recombinant plasmid pGFP--M, pGFP--HN, pGFP"-F and pGFP- -NP respectively.But expression was not proved in transfected cells and culture media analyzed by SDS-PAGE,indirected mmunity fluorescence and RT-PCR. The cloning and selection are undergoing now.
To investigate the possibility of construction of NDV-like particle in insect cell or out of cell, we sub-cloned successfully the M, F and HN gene of NDV into recombinant baculovirus transposition vector pFASTBACl, and transformed them into DH10BAC cells, by scanning plaque of white and blue and identification of PCR, the recombinant baculovirus shuttle vector Bacmid-M, Bacmid-F and Bacmid-HN were obtained. The recombinant baculovirus shuttle vector then were transfected into Sf9 insect cells respectively and the recombinant baculovirus rBacmid-M, rBacmid-F and rBacmid-HN were got. SDS-PAGE analysis showed that M and F gene have been expressed in Sf9 cells infected with recombinant baculoviruses.
Two kinds of expression system have been adopted to study the budding and assembly of ND-VLPs in this research. One is the mammalian cells expression system ,the proteins M, NP, F and HN expressed in mammalian cells can be used to study the detail interaction mechanism of1 NDV in cells; another is baculoviruses expression system with advantange of forgein protein expression in large quantity, so we can purify the expressed proteins and assembly ND-VLPs in vitro.
本研究首先构建了可能与ND-VLPs形成有关的NDV国内标准强毒F48E9株的结构基因M、F和HN的哺乳动物细胞重组表达载体pGFP-M、pGFP-HN和pGFP-F，经过限制性内切酶分析、质粒PCR鉴定和序列分析，证明我们获得了相应基因的哺乳动物细胞重组表达载体阳性质粒。然后将pGFP~--M、pGFP~--HN、pGFP~--F和pGFP~--NP共转染仓鼠肾细胞(BHK21)，72小时后收集共转染的细胞和上清应用电镜检测到了与NDV类似的颗粒，数量很少，结构也不典型，可能是共转染和表达效率较低的原因。为了解决这一问题，我们又将这些质粒分别转染BHK21，经过选择性培养基筛选后，获得了分别含有pGFP~--M、pGFP~--HN、pGFP~--NP和pGFP~--F重组表达质粒的细胞系，克隆筛选工作尚在进行中。对第一代转染的细胞培养上清和细胞应用SDS-PAGE、RT-PCR和间接免疫荧光检测，均未获得阳性结果。
同时，为了研究ND-VLPs在昆虫细胞中以及细胞外形成的可能性，我们将M、F和HN基因亚克隆到pFASTBAC1载体上，从而成功地构建了重组杆状病毒转座载体pFASTBAC1-M、pFASTBAC1-F和pFASTBAC1-HN，并分别转化到DH10BAC感受态细胞中，通过筛选获得了重组的杆状病毒穿梭载体Bacmid-M、Bacmid-F利Bacmid-HN。将重组杆状病毒穿梭载体分别转染Sf9细胞，经PCR检测获得了重组的杆状病毒。重组的杆状病毒分别感染Sf9细胞，SDS-PAGE检测到了M和F蛋白的表达，但没有检测到HN蛋白的表达。
本试验中我们构建了两种表达系统来研究ND-VLPs的形成和组装，不仅为揭示NDV的出芽机制、各基因及基因内不同功能区在病毒山芽、复制与组装过程中的作用，而且为进一步研究NDV的分子生物学特性，研制高效力的多价亚单位疫苗奠定了基础。
As a efficient investigation method, Virus-Like Particles (VLPs) technique has play an important role in the study of character, polymorphism, vaccine of many viruses. So the application of VLPs technique will help to discover the budding mechanism, develop novel, efficient and safe vaccine of NDV.
We constructed the mammalian cells expressing vector pGFP- -M, pGFP- -HN and pGFP- -F based on the structure gene of F48E9 strain of NDV,which is correlative with the formation of ND-VLPs.Then the recombinant plasmid pGFP- -M, pGFP- -HN, pGFP--F and pGFP--NP were co- transfected into BHK21 cells , some particles like NDV can be observed by electronic microscopy in collected 72h-post co-transfected cells and culture media,but we couldn't draw a conclusion that it's just ND-VLPs. To increase the efficiency of co-transfection, pGFP--IVK pGFP- -HN and pGFP'-F were transfected into BHK21 cells respectively, and screening with G-418 selective media, the result show that we have ot the stable cell line harbouring the recombinant plasmid pGFP--M, pGFP--HN, pGFP"-F and pGFP- -NP respectively.But expression was not proved in transfected cells and culture media analyzed by SDS-PAGE,indirected mmunity fluorescence and RT-PCR. The cloning and selection are undergoing now.
To investigate the possibility of construction of NDV-like particle in insect cell or out of cell, we sub-cloned successfully the M, F and HN gene of NDV into recombinant baculovirus transposition vector pFASTBACl, and transformed them into DH10BAC cells, by scanning plaque of white and blue and identification of PCR, the recombinant baculovirus shuttle vector Bacmid-M, Bacmid-F and Bacmid-HN were obtained. The recombinant baculovirus shuttle vector then were transfected into Sf9 insect cells respectively and the recombinant baculovirus rBacmid-M, rBacmid-F and rBacmid-HN were got. SDS-PAGE analysis showed that M and F gene have been expressed in Sf9 cells infected with recombinant baculoviruses.
Two kinds of expression system have been adopted to study the budding and assembly of ND-VLPs in this research. One is the mammalian cells expression system ,the proteins M, NP, F and HN expressed in mammalian cells can be used to study the detail interaction mechanism of1 NDV in cells; another is baculoviruses expression system with advantange of forgein protein expression in large quantity, so we can purify the expressed proteins and assembly ND-VLPs in vitro.
引文
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