Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt t...Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.展开更多
Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR...Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6, IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology. 2005;2(2):130-135.展开更多
基金supported by National High Technology Research and Development Program of China(863 program 2004AA215242)National Science Found for Distinguished Young Scholars from NSFC(No.39925031)Science and Technology commission of Shanghai municipality(024319112).
文摘Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.
基金Financial support for this work was provided by the National High Technology Research and Development Program of China(2004AA215242)by Science and Technology Commission of Shanghai Municipality(04XD14003).
文摘Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)can directly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptor TLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6, IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology. 2005;2(2):130-135.
