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
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
Background:Human leukocyte antigen(HLA)-DP is much less studied than other HLA class Ⅱ antigens,that is,HLA-DR and HLA-DQ,etc.However,the accumulating data have suggested the important roles of DP-restricted response...Background:Human leukocyte antigen(HLA)-DP is much less studied than other HLA class Ⅱ antigens,that is,HLA-DR and HLA-DQ,etc.However,the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer,allergy,and infectious disease.Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.Methods:To explore the potential cis-acting control elements involved in the tran-scriptional regulation of the HLA-DPA1/DPB1 gene,we performed the expression analysis using bacterial artificial chromosome(BAC)-based transgenic humanized mice in the C57BL/6 background,which carried the entire HLA-DP401 gene locus.We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice,and performed the analysis on the expres-sion pattern of HLA-DP401 and immunological responses in the model.Results:In this study,we screened and identified a BAC clone spanning the entire HLA-DP gene locus.DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals.Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene.Transgene copy numbers were determined by real-time PCR analysis.HLA-DP401 gene expression was analyzed at the mRNA and protein level.Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice.Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.Conclusions:We generated several BAC transgenic mice,and analyzed the expres-sion of HLA-DPA1/DPB1 in those mice.A model of S aureus-induced pneumonia in the HLA-DP401-H2-Aβ1^(-/-)humanized mice was further developed,and S aureus in-fection upregulated the HLA-DP401 expression in thymus of those humanized mice.These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.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National Science and Technology Major Project,Grant/Award Number:2017ZX10304402-001-006 and 2017ZX10304402-001-012Shanghai Professional Platform for High-level Biosafety Pathogenic Microorganism Detection,Grant/Award Number:18DZ2293000+1 种基金Scientific Research Projects of Shanghai Science and Technology Commission,Grant/Award Number:19140905300Shanghai Public Health Clinical Center General Program and the Start-on Funding,Grant/Award Number:KY-GW-2017-06,KY-GW-2018-11,KY-GW-2018-04,KY-GW-2019-11 and KY-GW-2019-19。
文摘Background:Human leukocyte antigen(HLA)-DP is much less studied than other HLA class Ⅱ antigens,that is,HLA-DR and HLA-DQ,etc.However,the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer,allergy,and infectious disease.Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.Methods:To explore the potential cis-acting control elements involved in the tran-scriptional regulation of the HLA-DPA1/DPB1 gene,we performed the expression analysis using bacterial artificial chromosome(BAC)-based transgenic humanized mice in the C57BL/6 background,which carried the entire HLA-DP401 gene locus.We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice,and performed the analysis on the expres-sion pattern of HLA-DP401 and immunological responses in the model.Results:In this study,we screened and identified a BAC clone spanning the entire HLA-DP gene locus.DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals.Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene.Transgene copy numbers were determined by real-time PCR analysis.HLA-DP401 gene expression was analyzed at the mRNA and protein level.Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice.Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.Conclusions:We generated several BAC transgenic mice,and analyzed the expres-sion of HLA-DPA1/DPB1 in those mice.A model of S aureus-induced pneumonia in the HLA-DP401-H2-Aβ1^(-/-)humanized mice was further developed,and S aureus in-fection upregulated the HLA-DP401 expression in thymus of those humanized mice.These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.
