To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette wit...To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.展开更多
为了研究家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)基因orf98的功能,通过λRed重组系统定点敲除BmNPVorf98基因,构建缺失型重组病毒Bm98-ko-Bacmid;以Bac-to-Bac系统补回BmNPVorf98基因,构建补回型重组病毒Bm98-re-B...为了研究家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)基因orf98的功能,通过λRed重组系统定点敲除BmNPVorf98基因,构建缺失型重组病毒Bm98-ko-Bacmid;以Bac-to-Bac系统补回BmNPVorf98基因,构建补回型重组病毒Bm98-re-Bacmid;将野生型病毒(wtBacmid)、缺失型病毒(Bm98-koBacmid)和补回型病毒(Bm98-re-Bacmid)分别转染家蚕细胞BmN。病毒滴度检测结果显示,Bm98-ko-Bacmid可形成侵染性的病毒粒子,但数量显著降低(P<0.05).透射电子显微镜观察发现,Bm98-ko-Bacmid只产生游离的杆状病毒粒子,数量明显减少,而wtBacmid和Bm98-re-Bacmid产生大量具有囊膜结构的成熟病毒粒子。荧光定量聚合酶链反应分析结果表明,BmNPVorf98基因缺失对BmNPV病毒复制没有影响,而早期基因lef3、晚期基因vp39和极晚期基因p10的转录水平显著降低(P<0.05)。综上所述,BmNPVorf98基因对病毒复制是非必需的,但显著影响病毒的繁殖速度和包装(P<0.05);对病毒各个时期的基因转录也具有重要影响。展开更多
Bm59是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)的核心基因之一。利用λRed重组技术和Bac-to-Bac系统构建野生型病毒wt Bacmid-polh-egfp、缺失型病毒Bm59ko-Bacmid-polh-egfp以及补回型病毒Bm59re-Bacmidpolh-eg...Bm59是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)的核心基因之一。利用λRed重组技术和Bac-to-Bac系统构建野生型病毒wt Bacmid-polh-egfp、缺失型病毒Bm59ko-Bacmid-polh-egfp以及补回型病毒Bm59re-Bacmidpolh-egfp,并分别转染家蚕卵巢培养细胞Bm N,研究Bm59基因在病毒侵染、增殖和组装中的功能。对病毒滴度的检测结果表明3种类型病毒都能产生有活力的子代病毒并使细胞感染发病,但Bm59ko-Bacmid-polh-egfp在各个时相的滴度值均显著低于其他2种类型病毒(P<0.05)。电子显微镜下观察Bm59ko-Bacmid-polh-egfp转染的细胞中只有少量细长的杆状病毒粒子,而其他2种类型病毒转染细胞后则能产生大量具有囊膜包裹的成熟病毒粒子。qRT-PCR检测结果显示,Bm59缺失型病毒基因组DNA的复制能力显著降低(P<0.05),早期基因lef-3、晚期基因vp39和极晚期基因p10的转录水平显著低于野生型病毒(P<0.05)。综上所述,Bm59基因虽然是家蚕核型多角体病毒复制的非必需基因,但会显著影响病毒的增殖速度和病毒粒子的组装,对病毒各个时期的基因转录水平具有下调作用。展开更多
基金973 (2003CB114202) Programme Strategic Scientific Alliances between China and the Netherlands (2004CB720404) National Natural Fundation of China project (30630002)
文摘To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.
文摘为了研究家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)基因orf98的功能,通过λRed重组系统定点敲除BmNPVorf98基因,构建缺失型重组病毒Bm98-ko-Bacmid;以Bac-to-Bac系统补回BmNPVorf98基因,构建补回型重组病毒Bm98-re-Bacmid;将野生型病毒(wtBacmid)、缺失型病毒(Bm98-koBacmid)和补回型病毒(Bm98-re-Bacmid)分别转染家蚕细胞BmN。病毒滴度检测结果显示,Bm98-ko-Bacmid可形成侵染性的病毒粒子,但数量显著降低(P<0.05).透射电子显微镜观察发现,Bm98-ko-Bacmid只产生游离的杆状病毒粒子,数量明显减少,而wtBacmid和Bm98-re-Bacmid产生大量具有囊膜结构的成熟病毒粒子。荧光定量聚合酶链反应分析结果表明,BmNPVorf98基因缺失对BmNPV病毒复制没有影响,而早期基因lef3、晚期基因vp39和极晚期基因p10的转录水平显著降低(P<0.05)。综上所述,BmNPVorf98基因对病毒复制是非必需的,但显著影响病毒的繁殖速度和包装(P<0.05);对病毒各个时期的基因转录也具有重要影响。
