两种快速构建重组杆状病毒的方法的建立及棉铃虫病毒基因组中orf107和orf135的鉴定和初步功能分析
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摘要
棉铃虫单粒包埋核多角体病毒(HaSNPV)的基因组全序列共有135个ORF，其中20个ORF是为棉铃虫病毒所特有的。为了快速地了解这些ORF在棉铃虫病毒的生命活动过程中的作用，我们发展了两套快速的构建突变型重组杆状病毒的方法，这些方法的建立为杆状病毒功能基因组学的研究提供了强有力的工具。利用这两套方法，我们获得了棉铃虫病毒基因组中的两个特有基因orf135和off107的突变重组子，结合表达和结构分析等对这两个基因进行了初步功能鉴定。
第一种快速构建重组杆状病毒的方法是在大肠杆菌中利用线性片断进行同源重组的方法。该方法建立在入噬菌体Red重组系统及杆状病毒的细菌人工染色体Bacmid的基础上。我们将棉铃虫病毒的细菌人工染色体HaBacHZ8转化进入含有入噬菌体Red重组系统的大肠杆菌BW25113(pKD46)中，再将同源重组线性片断导入，在Red重组系统的介导下，线性片断与HaBacHZ8发生同源重组，获得重组杆状病毒。该方法的重组过程发生在原核系统，避免了传统的共转染／空板纯化重组方法的耗时耗材。在此基础上，我们对该方法进行了进一步拓展，发展出直接用PCR产物构建同源重组杆状病毒的方法，使重组病毒的构建更为便捷。
第二种重组病毒的构建方法是利用转座子系统构建了棉铃虫病毒细菌人工染色体HaBacHZ8的随机插入突变文库。我们以TnMax13为转座原构建了含有1300个突变子的转座子随机插入突变文库。利用三轮PCR的筛选程序和特定PER产物相适合的鉴定原则，可对转座突变子进行定向筛选。多个基因突变子的筛选均证明了HaBacHZ8随机转座子突变子文库的构建是成功的。
推测的orf107的编码产物是一种高度疏水性的蛋白质，在原核系统中难于表达。为了检测orf107的在细胞中的表达情况，对orf107实行了截短型的原核表达，以截短型的表达产物为抗原免疫家兔制备了抗血清。以纯化的抗血清对感染HaSNPV的HzAM1细胞进行Wes tern blot检测，发现了抗血清可与大于170kDa的一种蛋白质产生免疫反应。对orf107的缺失突变子HaBacHS1和转座突变子HaBacHS2的研究表明，orf107是HaSNPV感染复制的非必需基因。
orf135的原核表达可以产生一种28kDa的蛋白质，以其为抗原制备的抗ORF135的抗血清检测到HaSNPV感染HzAM1后24h-72h间表达出一种26kDa的蛋白质。对orf135的缺失突变子的研究表明orf135在病毒的生命活动过程中起重要作用。
The genome of Heliocoverpa armigera single nucleocapsid polyhedrovirus consists of 135 ORFs, 20 of which are unique to the virus. In order to understand the function of the ORFs, two methods of generating recombinant mutants were developed. The methods have become important tools for functional genomic research of HaSNPV. With these two methods, recombinant HaSNPVs with mutations in two unique genes, orf707 and orf735, were constructed and their biological activities were studied. Together with the structure and expression analysis, the putative functions of orf707 and orf735 were characterized.
The method of linear homologous recombinant in Escherichia coli was founded on the basis the phage Red system and the bacterial artificial choromosome (BAC). Firstly, the BAC of HaSNPV, HaBacHZ8, was transformed into E. coli strain BW25113 (pKD46) containing the phage Red system. Then a linear fragment with homologue arms of the target gene was transformed into the bacterial, and the homologue recombination occurred with the help of the recombinase. As the recombination process takes place in the prokaryotic system, it greatly reduces the time and expense of the traditional method of co-transfection/plaque purification. This method was further improved by constructing linear fragment with PCR products, which simplified the process significantly.
Another method is to make a random insertion library. A random mutant library of HaBacHZ8 containing 1300 clones was constructed with transpon TnMaxl3. Target screening was established by primary selection of three rounds of PCR and further identification with PCR and sequencing. The successful screenings of many target mutants have demonstrated that the library was successful.
The truncated orf707 was cloned and expressed as a 20kDa protein in E coli. The antiserum was derived by immunizing rabbits with this protein. Western blot analysis using the antiserum revealed a protein larger than 170kDa in the HaSNPV infected cells. The studies of orf707 deletion mutant HaBacHSl and insertion mutant HaBacHS2 indicated that or/707 is an un-necessary gene for HaSNPV infection.
The orf135 was expressed as a 28kDa protein by prokaryotic expression vector, and the antiserum was produced in rabbits. Western blot analysis revealed a specific protein band with a size of 26kDa from the HaSNPV-infected HzAMl cells at 24h to 72h post infection. The study of orf135 deletion mutant indicated that orf135 plays an important role in the life cycle of HaSNPV.
第一种快速构建重组杆状病毒的方法是在大肠杆菌中利用线性片断进行同源重组的方法。该方法建立在入噬菌体Red重组系统及杆状病毒的细菌人工染色体Bacmid的基础上。我们将棉铃虫病毒的细菌人工染色体HaBacHZ8转化进入含有入噬菌体Red重组系统的大肠杆菌BW25113(pKD46)中，再将同源重组线性片断导入，在Red重组系统的介导下，线性片断与HaBacHZ8发生同源重组，获得重组杆状病毒。该方法的重组过程发生在原核系统，避免了传统的共转染／空板纯化重组方法的耗时耗材。在此基础上，我们对该方法进行了进一步拓展，发展出直接用PCR产物构建同源重组杆状病毒的方法，使重组病毒的构建更为便捷。
第二种重组病毒的构建方法是利用转座子系统构建了棉铃虫病毒细菌人工染色体HaBacHZ8的随机插入突变文库。我们以TnMax13为转座原构建了含有1300个突变子的转座子随机插入突变文库。利用三轮PCR的筛选程序和特定PER产物相适合的鉴定原则，可对转座突变子进行定向筛选。多个基因突变子的筛选均证明了HaBacHZ8随机转座子突变子文库的构建是成功的。
推测的orf107的编码产物是一种高度疏水性的蛋白质，在原核系统中难于表达。为了检测orf107的在细胞中的表达情况，对orf107实行了截短型的原核表达，以截短型的表达产物为抗原免疫家兔制备了抗血清。以纯化的抗血清对感染HaSNPV的HzAM1细胞进行Wes tern blot检测，发现了抗血清可与大于170kDa的一种蛋白质产生免疫反应。对orf107的缺失突变子HaBacHS1和转座突变子HaBacHS2的研究表明，orf107是HaSNPV感染复制的非必需基因。
orf135的原核表达可以产生一种28kDa的蛋白质，以其为抗原制备的抗ORF135的抗血清检测到HaSNPV感染HzAM1后24h-72h间表达出一种26kDa的蛋白质。对orf135的缺失突变子的研究表明orf135在病毒的生命活动过程中起重要作用。
The genome of Heliocoverpa armigera single nucleocapsid polyhedrovirus consists of 135 ORFs, 20 of which are unique to the virus. In order to understand the function of the ORFs, two methods of generating recombinant mutants were developed. The methods have become important tools for functional genomic research of HaSNPV. With these two methods, recombinant HaSNPVs with mutations in two unique genes, orf707 and orf735, were constructed and their biological activities were studied. Together with the structure and expression analysis, the putative functions of orf707 and orf735 were characterized.
The method of linear homologous recombinant in Escherichia coli was founded on the basis the phage Red system and the bacterial artificial choromosome (BAC). Firstly, the BAC of HaSNPV, HaBacHZ8, was transformed into E. coli strain BW25113 (pKD46) containing the phage Red system. Then a linear fragment with homologue arms of the target gene was transformed into the bacterial, and the homologue recombination occurred with the help of the recombinase. As the recombination process takes place in the prokaryotic system, it greatly reduces the time and expense of the traditional method of co-transfection/plaque purification. This method was further improved by constructing linear fragment with PCR products, which simplified the process significantly.
Another method is to make a random insertion library. A random mutant library of HaBacHZ8 containing 1300 clones was constructed with transpon TnMaxl3. Target screening was established by primary selection of three rounds of PCR and further identification with PCR and sequencing. The successful screenings of many target mutants have demonstrated that the library was successful.
The truncated orf707 was cloned and expressed as a 20kDa protein in E coli. The antiserum was derived by immunizing rabbits with this protein. Western blot analysis using the antiserum revealed a protein larger than 170kDa in the HaSNPV infected cells. The studies of orf707 deletion mutant HaBacHSl and insertion mutant HaBacHS2 indicated that or/707 is an un-necessary gene for HaSNPV infection.
The orf135 was expressed as a 28kDa protein by prokaryotic expression vector, and the antiserum was produced in rabbits. Western blot analysis revealed a specific protein band with a size of 26kDa from the HaSNPV-infected HzAMl cells at 24h to 72h post infection. The study of orf135 deletion mutant indicated that orf135 plays an important role in the life cycle of HaSNPV.
引文
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