目的：克隆变形链球菌葡聚糖结合蛋白C基因(Glucan-binding protein C gene,gbpC)编码序列的特异片段,并在大肠杆菌中实现原核表达。方法：根据gbpC序列设计并合成一对寡核苷酸引物,PCR扩增位于gbpC编码序列1342 bp～1794 bp间的一段特...目的：克隆变形链球菌葡聚糖结合蛋白C基因(Glucan-binding protein C gene,gbpC)编码序列的特异片段,并在大肠杆菌中实现原核表达。方法：根据gbpC序列设计并合成一对寡核苷酸引物,PCR扩增位于gbpC编码序列1342 bp～1794 bp间的一段特异片段,扩增产物经回收、酶切后,定向插入克隆载体puc18中,连接产物转化感受态大肠杆菌DH5α,挑选阳性克隆,鉴定后进行序列测定。将克隆获得的约0．45 kb的gbpC基因特异片段经EcoRI/SalI双酶切后。定向插入pGEX-4T-1中,构建pGEX-4T-1/gbpCE原核融合表达载体,转化感受态大肠杆菌DH5α,挑选阳性克隆,酶切及PCR鉴定后,异丙基硫代-β-D-半乳糖苷(Isopropyl-β-D-thiogalactoside,IPTG)诱导表达融合蛋白GST—GbpC^E,SDS—PAGE检测表达产物。结果：测序结果与Sato等报道的序列一致；原核表达后,在SDS—PAGE凝胶上出现一条新生蛋白条带,其相对分子量约43000,与预计大小相符合。结论：成功克隆并表达gbpC特异片段,获得了融合蛋白GST-GbpC^E,为制备GbpC抗体打下了基础。展开更多
目的探索 C 端结构变异为亮氨酸-脯氨酸-任意氨基酸-丙氨酸-甘氨酸(LPXAG)的葡聚糖结合蛋白 C(glucan binding protein C,GbpC)是否在变形链球菌 UA159的细胞壁上。方法用PCR 法扩增变形链球菌 UA159 GbpC C 端的编码基因片段,推测和分...目的探索 C 端结构变异为亮氨酸-脯氨酸-任意氨基酸-丙氨酸-甘氨酸(LPXAG)的葡聚糖结合蛋白 C(glucan binding protein C,GbpC)是否在变形链球菌 UA159的细胞壁上。方法用PCR 法扩增变形链球菌 UA159 GbpC C 端的编码基因片段,推测和分析 C 端氨基酸序列组成;分别提取上清蛋白、胞内蛋白和胞壁蛋白,用 Western blot 法在变形链球菌 UA159各成分中检测 GbpC;用免疫金标直接观察 GbpC 的表达位置;应激条件下进行多聚糖依赖黏附实验。结果变形链球菌 UA159GbpC C 端亮氨酸-脯氨酸-任意氨基酸-苏氨酸-甘氨酸(LPXTG)的结构变异为 LPXAG;免疫印迹可以在细菌的壁蛋白中检测到 GbpC;电镜下可以观察到金颗粒围绕细菌细胞壁聚集;变形链球菌 UA159在应激条件下表现为多糖依赖黏附实验阳性。结论 C 端为 LPXAG 的 GbpC 仍然锚定在细菌的细胞壁上。展开更多
Bacteria contamination in Auricularia auricular culture bags reduces yield and increases the risk of food safety.In this study,5 species of bacteria,mainly gram-positive bacteria including three species of Bacillus sp...Bacteria contamination in Auricularia auricular culture bags reduces yield and increases the risk of food safety.In this study,5 species of bacteria,mainly gram-positive bacteria including three species of Bacillus spp.,Arthrobacter arilaitensis and Staphylococcus warneri,were isolated and identifi ed from bacteria-contaminated A.auricular culture bags.An in silico predicted antimicrobial peptide from theβ-1,3-glucan-binding protein sequence of Penaeus vannamei,designated Pv GBP2(FLKLGRKSRYGMLKL),was screened and its antibacterial effect and mechanism of action on the isolated Bacillus spp.explored.The minimal inhibitory concentrations(MIC)of Pv GBP2 on Bacillus spp.were 15.6–31.25μg/m L.Peptide Pv GBP2 could inhibit Bacillus subtilis in A.auricular culture bags to maintain growth and yield of A.auricular.Transmission electron microscopy(TEM)revealed that Pv GBP2 kills bacteria by perforating the cell wall,destroying membrane integrity and resulting in the leakage of intracellular solutes.In addition,Pv GBP2 inhibits biofi lm formation by B.subtilis by 90.6%at 1×MIC.Thus,peptide Pv GBP2 could be potentially applied as an antibacterial agent to control bacterial infection of A.auricular cultivation and the spread of foodborne pathogens.展开更多
文摘目的：克隆变形链球菌葡聚糖结合蛋白C基因(Glucan-binding protein C gene,gbpC)编码序列的特异片段,并在大肠杆菌中实现原核表达。方法：根据gbpC序列设计并合成一对寡核苷酸引物,PCR扩增位于gbpC编码序列1342 bp～1794 bp间的一段特异片段,扩增产物经回收、酶切后,定向插入克隆载体puc18中,连接产物转化感受态大肠杆菌DH5α,挑选阳性克隆,鉴定后进行序列测定。将克隆获得的约0．45 kb的gbpC基因特异片段经EcoRI/SalI双酶切后。定向插入pGEX-4T-1中,构建pGEX-4T-1/gbpCE原核融合表达载体,转化感受态大肠杆菌DH5α,挑选阳性克隆,酶切及PCR鉴定后,异丙基硫代-β-D-半乳糖苷(Isopropyl-β-D-thiogalactoside,IPTG)诱导表达融合蛋白GST—GbpC^E,SDS—PAGE检测表达产物。结果：测序结果与Sato等报道的序列一致；原核表达后,在SDS—PAGE凝胶上出现一条新生蛋白条带,其相对分子量约43000,与预计大小相符合。结论：成功克隆并表达gbpC特异片段,获得了融合蛋白GST-GbpC^E,为制备GbpC抗体打下了基础。
文摘目的探索 C 端结构变异为亮氨酸-脯氨酸-任意氨基酸-丙氨酸-甘氨酸(LPXAG)的葡聚糖结合蛋白 C(glucan binding protein C,GbpC)是否在变形链球菌 UA159的细胞壁上。方法用PCR 法扩增变形链球菌 UA159 GbpC C 端的编码基因片段,推测和分析 C 端氨基酸序列组成;分别提取上清蛋白、胞内蛋白和胞壁蛋白,用 Western blot 法在变形链球菌 UA159各成分中检测 GbpC;用免疫金标直接观察 GbpC 的表达位置;应激条件下进行多聚糖依赖黏附实验。结果变形链球菌 UA159GbpC C 端亮氨酸-脯氨酸-任意氨基酸-苏氨酸-甘氨酸(LPXTG)的结构变异为 LPXAG;免疫印迹可以在细菌的壁蛋白中检测到 GbpC;电镜下可以观察到金颗粒围绕细菌细胞壁聚集;变形链球菌 UA159在应激条件下表现为多糖依赖黏附实验阳性。结论 C 端为 LPXAG 的 GbpC 仍然锚定在细菌的细胞壁上。
基金sponsored by National Key R&D Program of China(2020YFD09009)。
文摘Bacteria contamination in Auricularia auricular culture bags reduces yield and increases the risk of food safety.In this study,5 species of bacteria,mainly gram-positive bacteria including three species of Bacillus spp.,Arthrobacter arilaitensis and Staphylococcus warneri,were isolated and identifi ed from bacteria-contaminated A.auricular culture bags.An in silico predicted antimicrobial peptide from theβ-1,3-glucan-binding protein sequence of Penaeus vannamei,designated Pv GBP2(FLKLGRKSRYGMLKL),was screened and its antibacterial effect and mechanism of action on the isolated Bacillus spp.explored.The minimal inhibitory concentrations(MIC)of Pv GBP2 on Bacillus spp.were 15.6–31.25μg/m L.Peptide Pv GBP2 could inhibit Bacillus subtilis in A.auricular culture bags to maintain growth and yield of A.auricular.Transmission electron microscopy(TEM)revealed that Pv GBP2 kills bacteria by perforating the cell wall,destroying membrane integrity and resulting in the leakage of intracellular solutes.In addition,Pv GBP2 inhibits biofi lm formation by B.subtilis by 90.6%at 1×MIC.Thus,peptide Pv GBP2 could be potentially applied as an antibacterial agent to control bacterial infection of A.auricular cultivation and the spread of foodborne pathogens.
