Objective ： To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit （LT-B） gene and generate LT-B transgenic tobacco plants. Methods： The LT-B coding sequence was amp...Objective ： To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit （LT-B） gene and generate LT-B transgenic tobacco plants. Methods： The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus （CaMV） 35S promoter. The tobacco plants （Nicotiana tobacum L. Cuttivar Xanthi） were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Resuits： LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3. 36-10. 56 ng/mg total soluble tobacco leaf protein. Conclusion： The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the preven- tion of mucosaroute evading pathogen.展开更多
Human papillomavirus （HPV）, mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated c...Human papillomavirus （HPV）, mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated cervical cancer.展开更多
目的原核表达、纯化大肠杆菌不耐热肠毒素B亚单位(Heat-labile enterotoxin B subunit,LTB),并评价其黏膜免疫的佐剂作用。方法从产毒素大肠杆菌44815菌株中扩增出LTB基因,克隆至质粒pET-32a(+)中,构建重组原核表达质粒pET-32a(+)-LTB,...目的原核表达、纯化大肠杆菌不耐热肠毒素B亚单位(Heat-labile enterotoxin B subunit,LTB),并评价其黏膜免疫的佐剂作用。方法从产毒素大肠杆菌44815菌株中扩增出LTB基因,克隆至质粒pET-32a(+)中,构建重组原核表达质粒pET-32a(+)-LTB,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达,表达的LTB经纯化后进行神经节苷脂GM1结合活性及黏膜免疫佐剂活性鉴定。结果重组表达质粒经双酶切及测序鉴定证明构建正确;重组LTB主要为可溶性表达,表达量约占菌体总蛋白的15%;纯化的LTB纯度可达95%以上,可与兔抗CTB血清发生特异性反应,且具有GM1结合活性及良好的黏膜免疫佐剂活性。结论在大肠杆菌中成功表达了重组LTB蛋白,纯化的LTB具有良好的黏膜免疫佐剂活性。展开更多
基金Supported by the National Natural Science Foundation ofChina (No. 30070848)
文摘Objective ： To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit （LT-B） gene and generate LT-B transgenic tobacco plants. Methods： The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus （CaMV） 35S promoter. The tobacco plants （Nicotiana tobacum L. Cuttivar Xanthi） were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Resuits： LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3. 36-10. 56 ng/mg total soluble tobacco leaf protein. Conclusion： The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the preven- tion of mucosaroute evading pathogen.
文摘Human papillomavirus （HPV）, mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated cervical cancer.
文摘目的原核表达、纯化大肠杆菌不耐热肠毒素B亚单位(Heat-labile enterotoxin B subunit,LTB),并评价其黏膜免疫的佐剂作用。方法从产毒素大肠杆菌44815菌株中扩增出LTB基因,克隆至质粒pET-32a(+)中,构建重组原核表达质粒pET-32a(+)-LTB,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达,表达的LTB经纯化后进行神经节苷脂GM1结合活性及黏膜免疫佐剂活性鉴定。结果重组表达质粒经双酶切及测序鉴定证明构建正确;重组LTB主要为可溶性表达,表达量约占菌体总蛋白的15%;纯化的LTB纯度可达95%以上,可与兔抗CTB血清发生特异性反应,且具有GM1结合活性及良好的黏膜免疫佐剂活性。结论在大肠杆菌中成功表达了重组LTB蛋白,纯化的LTB具有良好的黏膜免疫佐剂活性。
