微生物污染是饮片外源性污染的主要形式之一,也是影响人体健康的重要途径。对近10年来饮片微生物污染及典型菌研究进展进行综述,结果显示目前我国饮片微生物污染较为严重,不同品种、来源及炮制方法饮片间微生物污染存在较大差异,耐热菌...微生物污染是饮片外源性污染的主要形式之一,也是影响人体健康的重要途径。对近10年来饮片微生物污染及典型菌研究进展进行综述,结果显示目前我国饮片微生物污染较为严重,不同品种、来源及炮制方法饮片间微生物污染存在较大差异,耐热菌及耐胆盐革兰阴性菌污染率高,其他控制菌检出率较低。污染典型菌的分离鉴定研究主要集中于耐热菌及革兰阴性杆菌,典型耐热菌主要是来源于土壤及其他外环境的芽胞杆菌属,少数分离菌株对人类有致病报道;分离的典型革兰阴性杆菌70%以上菌株有致病报道,以阴沟肠杆菌为常见分离菌。近年来,一些新的污染典型菌检测方法逐步被应用,如API、Vitek2 Compact鉴定系统、FRIT、MALDI-TOF/MS、核糖体分型技术及16 S rRNA宏基因组高通量测序技术等,这些方法较传统方法更灵敏、快捷和准确。展开更多
AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion ...AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.展开更多
Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, a...Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits. The isolates are Gram negative, rod-shaped bacteria, their optimal temperature and pH value for growth are 45-50℃ and 2.5-3.5 respectively. They are autotrophic and used elemental sulfur, sodium thiosulfate and potassium tetrathionate as electron donor, while a little glucose stimulated their growth. 16S rDNA sequences analysis reveals that the strains are phylogenetically clustered to Acidithiobacillus caldus.展开更多
文摘微生物污染是饮片外源性污染的主要形式之一,也是影响人体健康的重要途径。对近10年来饮片微生物污染及典型菌研究进展进行综述,结果显示目前我国饮片微生物污染较为严重,不同品种、来源及炮制方法饮片间微生物污染存在较大差异,耐热菌及耐胆盐革兰阴性菌污染率高,其他控制菌检出率较低。污染典型菌的分离鉴定研究主要集中于耐热菌及革兰阴性杆菌,典型耐热菌主要是来源于土壤及其他外环境的芽胞杆菌属,少数分离菌株对人类有致病报道;分离的典型革兰阴性杆菌70%以上菌株有致病报道,以阴沟肠杆菌为常见分离菌。近年来,一些新的污染典型菌检测方法逐步被应用,如API、Vitek2 Compact鉴定系统、FRIT、MALDI-TOF/MS、核糖体分型技术及16 S rRNA宏基因组高通量测序技术等,这些方法较传统方法更灵敏、快捷和准确。
文摘AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.
基金Project (50321402) supported by the National Natural Science Foundation of China Project(2004CB619204) supported by the State Basic Research Development Program of China Project (DY105-02-04-05) supported by the China Ocean Mineral Resources Research and Development Association
文摘Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits. The isolates are Gram negative, rod-shaped bacteria, their optimal temperature and pH value for growth are 45-50℃ and 2.5-3.5 respectively. They are autotrophic and used elemental sulfur, sodium thiosulfate and potassium tetrathionate as electron donor, while a little glucose stimulated their growth. 16S rDNA sequences analysis reveals that the strains are phylogenetically clustered to Acidithiobacillus caldus.
