杆状病毒是主要对昆虫显示病原性的一类病毒,可感染600多种昆虫,是自然生态体系统中调节虫口密度的主要生物因子之一。它以大的棒状病毒粒子、内含分子量为28～60kb 的闭环超螺旋双链 DNA 基因组的特征。分类上将杆状病毒种分为3个亚组...杆状病毒是主要对昆虫显示病原性的一类病毒,可感染600多种昆虫,是自然生态体系统中调节虫口密度的主要生物因子之一。它以大的棒状病毒粒子、内含分子量为28～60kb 的闭环超螺旋双链 DNA 基因组的特征。分类上将杆状病毒种分为3个亚组:A 亚组的核多角体病毒,以多角体蛋白结晶基质包涵许多有囊膜的核衣壳形成多角体为特征;苜蓿尺蠖核多角体病毒(AcNPV)和家蚕核多角体病毒(BmNPV)分别为多粒包埋型和单粒包埋型为代表种,B展开更多
p30蛋白是非洲猪瘟病毒(ASFV)免疫原性最强的结构蛋白之一,由CP204L基因编码。本研究利用Bac-to-Bac昆虫杆状病毒真核表达系统将ASFV中CP204L插入p Fast Bac1载体下游,将形成的重组质粒p Fast Bac-p30以转座子的形式插入到DH10Bac中的...p30蛋白是非洲猪瘟病毒(ASFV)免疫原性最强的结构蛋白之一,由CP204L基因编码。本研究利用Bac-to-Bac昆虫杆状病毒真核表达系统将ASFV中CP204L插入p Fast Bac1载体下游,将形成的重组质粒p Fast Bac-p30以转座子的形式插入到DH10Bac中的杆状病毒穿梭质粒(Bacmid)中,筛选出重组Bacmid-p30后,将Bacmid-p30转染Sf9细胞,并继续传代3次,获得重组杆状病毒,命名为r Bac-p30。分别通过间接免疫荧光(IFA)和免疫印迹(Western blot)鉴定了ASFV阳性血清可特异性识别Sf9细胞中表达的p30。以此真核表达p30蛋白包被ELISA板,可区分ASFV阴阳性血清。以上结果表明,本研究初步建立了检测ASFV血清抗体的间接ELISA方法,为后续建立非洲猪瘟抗体检测方法和p30亚单位疫苗研究奠定基础。展开更多
The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian...The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian sheep blowfly Lucilia cuprina(LcEcR). pSXIVVI +X3/2 baculovirus transfer vector was chosen for a 2.8kb LcEcR cDNA subcloning since pSXIVVI +X3/2 contains an efficient translational initiation signal (ATG) and it allows the LcEcR cDNA fusion to N-terminal codons in the correct reading frame. The resulting transfer plasmid pSXIVVI +X3-LcEcR was cotransfected into BT1-Tn-5B1-4 cells with the parental virus TnNPV-SVI -G minus polyhedral inclusion body, which expresses β-galactosidase gene. After 3～4 runs of plaque purification, three TnNPV-LcEcR clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters. These three TnNPV-LcEcR clones all showed white phenotype when stained with X-gal. Western blot analysis showed 2～3 specific polypeptides with molecular weight ranging from 70～90kD. Three TnNPV-LcEcR clones expressed different level of LcEcR polypeptides in BT1-Tn-5B1-4 cells. The TnNPV-LcEcR-1 clone expressed the highest level of LcEcR polypeptides in BT1-Tn-5B1-4 cells 48～72h post infection.展开更多
文摘杆状病毒是主要对昆虫显示病原性的一类病毒,可感染600多种昆虫,是自然生态体系统中调节虫口密度的主要生物因子之一。它以大的棒状病毒粒子、内含分子量为28～60kb 的闭环超螺旋双链 DNA 基因组的特征。分类上将杆状病毒种分为3个亚组:A 亚组的核多角体病毒,以多角体蛋白结晶基质包涵许多有囊膜的核衣壳形成多角体为特征;苜蓿尺蠖核多角体病毒(AcNPV)和家蚕核多角体病毒(BmNPV)分别为多粒包埋型和单粒包埋型为代表种,B
文摘p30蛋白是非洲猪瘟病毒(ASFV)免疫原性最强的结构蛋白之一,由CP204L基因编码。本研究利用Bac-to-Bac昆虫杆状病毒真核表达系统将ASFV中CP204L插入p Fast Bac1载体下游,将形成的重组质粒p Fast Bac-p30以转座子的形式插入到DH10Bac中的杆状病毒穿梭质粒(Bacmid)中,筛选出重组Bacmid-p30后,将Bacmid-p30转染Sf9细胞,并继续传代3次,获得重组杆状病毒,命名为r Bac-p30。分别通过间接免疫荧光(IFA)和免疫印迹(Western blot)鉴定了ASFV阳性血清可特异性识别Sf9细胞中表达的p30。以此真核表达p30蛋白包被ELISA板,可区分ASFV阴阳性血清。以上结果表明,本研究初步建立了检测ASFV血清抗体的间接ELISA方法,为后续建立非洲猪瘟抗体检测方法和p30亚单位疫苗研究奠定基础。
文摘The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian sheep blowfly Lucilia cuprina(LcEcR). pSXIVVI +X3/2 baculovirus transfer vector was chosen for a 2.8kb LcEcR cDNA subcloning since pSXIVVI +X3/2 contains an efficient translational initiation signal (ATG) and it allows the LcEcR cDNA fusion to N-terminal codons in the correct reading frame. The resulting transfer plasmid pSXIVVI +X3-LcEcR was cotransfected into BT1-Tn-5B1-4 cells with the parental virus TnNPV-SVI -G minus polyhedral inclusion body, which expresses β-galactosidase gene. After 3～4 runs of plaque purification, three TnNPV-LcEcR clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters. These three TnNPV-LcEcR clones all showed white phenotype when stained with X-gal. Western blot analysis showed 2～3 specific polypeptides with molecular weight ranging from 70～90kD. Three TnNPV-LcEcR clones expressed different level of LcEcR polypeptides in BT1-Tn-5B1-4 cells. The TnNPV-LcEcR-1 clone expressed the highest level of LcEcR polypeptides in BT1-Tn-5B1-4 cells 48～72h post infection.
