以E.coliDH5α基因组为模板PCR扩增得到无信号肽的碱性磷酸酶基因片段,与胞内融合表达型T载体连接得到重组质粒,转化表达宿主E.coliBL21(DE3)和E.coliorigami(DE3),经0.1 mM IPTG诱导表达,超声破碎细胞后,SDS-PAGE分析可溶性,融合蛋白在...以E.coliDH5α基因组为模板PCR扩增得到无信号肽的碱性磷酸酶基因片段,与胞内融合表达型T载体连接得到重组质粒,转化表达宿主E.coliBL21(DE3)和E.coliorigami(DE3),经0.1 mM IPTG诱导表达,超声破碎细胞后,SDS-PAGE分析可溶性,融合蛋白在E.coliorigami(DE3)中的可溶蛋白含量比E.coliBL21(DE3)中高.融合蛋白的可溶部分经Ni2+螯合亲和纯化,纯化后E.coliorigami(DE3)中蛋白活性比E.coliBL21(DE3)中明显提高,达到1 614.3U/mg蛋白.展开更多
According to the reported sequence of \%Buthus martensii\% Karsch scorpion toxin gene (BmK IT 3),we synthesized two primers,which were complementary in a region.By the means of PCR,we got the gene.The gene was fused i...According to the reported sequence of \%Buthus martensii\% Karsch scorpion toxin gene (BmK IT 3),we synthesized two primers,which were complementary in a region.By the means of PCR,we got the gene.The gene was fused in expression vector pET-28a,which gave rise to a recombinant plasmid pET(IT 3 R).Then it was transformed into \%E.coli\% BL21 (DE 3).With IPTG induction,the gene was efficiently expressed.And the fusion product was soluble.展开更多
目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质...目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质粒重组,转化大肠杆菌,获得GVP蛋白阳性表达菌株,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,产物经Ni+-NTA琼脂糖凝胶层析纯化,获得目的蛋白GVP,DNA结合实验检测其功能。结果:成功合成出GVP基因,重组体在大肠杆菌BL21(DE3)中诱导获得高效表达,DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论:重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力,可用于食品病原微生物特定单链DNA分子的浓缩和分离。展开更多
文摘以E.coliDH5α基因组为模板PCR扩增得到无信号肽的碱性磷酸酶基因片段,与胞内融合表达型T载体连接得到重组质粒,转化表达宿主E.coliBL21(DE3)和E.coliorigami(DE3),经0.1 mM IPTG诱导表达,超声破碎细胞后,SDS-PAGE分析可溶性,融合蛋白在E.coliorigami(DE3)中的可溶蛋白含量比E.coliBL21(DE3)中高.融合蛋白的可溶部分经Ni2+螯合亲和纯化,纯化后E.coliorigami(DE3)中蛋白活性比E.coliBL21(DE3)中明显提高,达到1 614.3U/mg蛋白.
文摘According to the reported sequence of \%Buthus martensii\% Karsch scorpion toxin gene (BmK IT 3),we synthesized two primers,which were complementary in a region.By the means of PCR,we got the gene.The gene was fused in expression vector pET-28a,which gave rise to a recombinant plasmid pET(IT 3 R).Then it was transformed into \%E.coli\% BL21 (DE 3).With IPTG induction,the gene was efficiently expressed.And the fusion product was soluble.
文摘目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质粒重组,转化大肠杆菌,获得GVP蛋白阳性表达菌株,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,产物经Ni+-NTA琼脂糖凝胶层析纯化,获得目的蛋白GVP,DNA结合实验检测其功能。结果:成功合成出GVP基因,重组体在大肠杆菌BL21(DE3)中诱导获得高效表达,DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论:重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力,可用于食品病原微生物特定单链DNA分子的浓缩和分离。
