AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity ...AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine. METHODS： The TGF-β1 encoding epitope gene （the mature TGF-β1 from 78-109 amino acid residues, TGF-β1^32） was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β1 ^32 vector. The HBcAg gene fragments （encoding HBcAg from 1-71 and 89-144 amino acid residues） were amplified from PYTAI- HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1^32 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 Ugase. The fusion gene fragments HBc.Ag1-71-TGF-β1^32 HBcAg89-144 were recloned to pET28a（＋） and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a （＋）/ CTC was transformed and expressed in E. coli BL21 （DE3） under induction of IPTG. After purification with Ni^＋2 NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS： Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass （Mr） of the expressed product by pET28a （＋）/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine. CONCLUSION： A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.展开更多
将64只SD雄性大鼠随机分为对照组、模型组和中药干预组(500、1000 mg/kg),对照组大鼠灌注1.0 ml 0.9%生理盐水造模,模型组和中药干预组灌注等量的SiO_(2)悬浊液,其后模型组和对照组每天灌胃0.9%生理盐水,中药干预组每天分别灌胃500、100...将64只SD雄性大鼠随机分为对照组、模型组和中药干预组(500、1000 mg/kg),对照组大鼠灌注1.0 ml 0.9%生理盐水造模,模型组和中药干预组灌注等量的SiO_(2)悬浊液,其后模型组和对照组每天灌胃0.9%生理盐水,中药干预组每天分别灌胃500、1000 mg/kg抗肺纤中药颗粒。造模后第14天和第28天处死大鼠,进行肺组织病理学观察及肺部抗肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、抗转化生长因子-β1(TGF-β1)、Smad2/3蛋白和小鼠来源抗Ⅰ型胶原蛋白(Col-Ⅰ)的水平检测。结果表明,中药干预可抑制SiO_(2)诱导大鼠的肺组织TNF-α、IL-1β和TGF-β1水平增高及Col-Ⅰ沉积,减缓大鼠肺泡炎症及矽肺纤维化进展,干预作用可能与TGF-β1/Smad通路有关。展开更多
文摘AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine. METHODS： The TGF-β1 encoding epitope gene （the mature TGF-β1 from 78-109 amino acid residues, TGF-β1^32） was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β1 ^32 vector. The HBcAg gene fragments （encoding HBcAg from 1-71 and 89-144 amino acid residues） were amplified from PYTAI- HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1^32 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 Ugase. The fusion gene fragments HBc.Ag1-71-TGF-β1^32 HBcAg89-144 were recloned to pET28a（＋） and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a （＋）/ CTC was transformed and expressed in E. coli BL21 （DE3） under induction of IPTG. After purification with Ni^＋2 NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS： Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass （Mr） of the expressed product by pET28a （＋）/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine. CONCLUSION： A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.
文摘将64只SD雄性大鼠随机分为对照组、模型组和中药干预组(500、1000 mg/kg),对照组大鼠灌注1.0 ml 0.9%生理盐水造模,模型组和中药干预组灌注等量的SiO_(2)悬浊液,其后模型组和对照组每天灌胃0.9%生理盐水,中药干预组每天分别灌胃500、1000 mg/kg抗肺纤中药颗粒。造模后第14天和第28天处死大鼠,进行肺组织病理学观察及肺部抗肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、抗转化生长因子-β1(TGF-β1)、Smad2/3蛋白和小鼠来源抗Ⅰ型胶原蛋白(Col-Ⅰ)的水平检测。结果表明,中药干预可抑制SiO_(2)诱导大鼠的肺组织TNF-α、IL-1β和TGF-β1水平增高及Col-Ⅰ沉积,减缓大鼠肺泡炎症及矽肺纤维化进展,干预作用可能与TGF-β1/Smad通路有关。
