从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA。用PCR、酶切法鉴定T7噬菌体DNA的完整性。通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方...从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA。用PCR、酶切法鉴定T7噬菌体DNA的完整性。通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法。结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确。T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng(DNA)。展开更多
目的观察不同因素对消毒剂灭活噬菌体效果的影响。方法采用正交设计试验和悬液定量病毒灭活试验方法,对含氯消毒剂灭活T7噬菌体效果的影响因素进行了观察。结果消毒剂浓度、灭活时间、小牛血清含量、消毒剂浓度与小牛血清含量的交互作...目的观察不同因素对消毒剂灭活噬菌体效果的影响。方法采用正交设计试验和悬液定量病毒灭活试验方法,对含氯消毒剂灭活T7噬菌体效果的影响因素进行了观察。结果消毒剂浓度、灭活时间、小牛血清含量、消毒剂浓度与小牛血清含量的交互作用以及灭活时间与小牛血清含量的交互作用对T7噬菌体的灭活效果的影响均有显著性意义,其对结果的影响程度由大到小的顺序为:小牛血清含量>消毒剂浓度>灭活时间>灭活时间×小牛血清含量>消毒剂浓度×小牛血清含量。在清洁条件下用有效氯150 mg/L对悬液内大肠杆菌T7噬菌体作用1 m in可达到完全灭活,在加入有机物条件下用有效氯250 mg/L对悬液内该噬菌体作用5 m in灭活率仅为99.97%。结论在设计的4个因素对T7噬菌体灭活效果影响的试验中,有机物含量、消毒剂浓度、作用时间等3个单因子影响显著,有机物与消毒剂浓度之间和作用时间与有机物之间存在交互作用。展开更多
Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the indu...Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.展开更多
In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage dis...In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.展开更多
文摘从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA。用PCR、酶切法鉴定T7噬菌体DNA的完整性。通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法。结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确。T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng(DNA)。
文摘目的观察不同因素对消毒剂灭活噬菌体效果的影响。方法采用正交设计试验和悬液定量病毒灭活试验方法,对含氯消毒剂灭活T7噬菌体效果的影响因素进行了观察。结果消毒剂浓度、灭活时间、小牛血清含量、消毒剂浓度与小牛血清含量的交互作用以及灭活时间与小牛血清含量的交互作用对T7噬菌体的灭活效果的影响均有显著性意义,其对结果的影响程度由大到小的顺序为:小牛血清含量>消毒剂浓度>灭活时间>灭活时间×小牛血清含量>消毒剂浓度×小牛血清含量。在清洁条件下用有效氯150 mg/L对悬液内大肠杆菌T7噬菌体作用1 m in可达到完全灭活,在加入有机物条件下用有效氯250 mg/L对悬液内该噬菌体作用5 m in灭活率仅为99.97%。结论在设计的4个因素对T7噬菌体灭活效果影响的试验中,有机物含量、消毒剂浓度、作用时间等3个单因子影响显著,有机物与消毒剂浓度之间和作用时间与有机物之间存在交互作用。
基金Supported by the National Natural Science Foundation of China(No.30671852)the Open Research Fund Program of the State Key Laboratory of Virology of China(Nos.2010009, 2007007) the Research Fund of the Key Laboratory of Department of Education of Liaoning Province, China(No.2009S043)
文摘Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.
文摘In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.
