目的 :利用 Bac- to- Bac表达系统高效表达成熟的胰岛素样生长因子 (IGF- )。方法 :首先构建含 IGF- c DNA的重组供体质粒 p Fast Bac1,然后重组 p Fast Bac1所含的转位元件 mini- Tn7,在感受态细胞 DH10 Bac内转位到粘粒上的连接位...目的 :利用 Bac- to- Bac表达系统高效表达成熟的胰岛素样生长因子 (IGF- )。方法 :首先构建含 IGF- c DNA的重组供体质粒 p Fast Bac1,然后重组 p Fast Bac1所含的转位元件 mini- Tn7,在感受态细胞 DH10 Bac内转位到粘粒上的连接位点mini- att Tn7构成重组粘粒 ,重组粘粒转染昆虫细胞 Sf9产生重组杆状病毒 ,重组杆状病毒进行感染 Sf9细胞。结果 :琼脂糖凝胶分析显示重组供体质粒 p Fast Bac1的成功构建 ;琼脂糖凝胶分析重组粘粒的 PCR产物表明已获得重组粘粒 ;十二烷基磺酸钠(SDS) - PAGE和 Western Blotting显示 7KD左右蛋白质条带的出现。结论 :利用 Bac- to- Bac表达系统成功表达具有免疫学活性的 IGF- ,可用于临床疾病的诊治。展开更多
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
In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper a...In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.展开更多
文摘目的 :利用 Bac- to- Bac表达系统高效表达成熟的胰岛素样生长因子 (IGF- )。方法 :首先构建含 IGF- c DNA的重组供体质粒 p Fast Bac1,然后重组 p Fast Bac1所含的转位元件 mini- Tn7,在感受态细胞 DH10 Bac内转位到粘粒上的连接位点mini- att Tn7构成重组粘粒 ,重组粘粒转染昆虫细胞 Sf9产生重组杆状病毒 ,重组杆状病毒进行感染 Sf9细胞。结果 :琼脂糖凝胶分析显示重组供体质粒 p Fast Bac1的成功构建 ;琼脂糖凝胶分析重组粘粒的 PCR产物表明已获得重组粘粒 ;十二烷基磺酸钠(SDS) - PAGE和 Western Blotting显示 7KD左右蛋白质条带的出现。结论 :利用 Bac- to- Bac表达系统成功表达具有免疫学活性的 IGF- ,可用于临床疾病的诊治。
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.
基金supported by the National Nature Science Foundation of China (31370188)Shenzhen Municipal Development and Reform Commission (Shenzhen Research and Development Center,Code:[2012]318)
文摘In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.
