目的:筛查变应原并明确蒿属植物种属鉴定,深入分析野艾蒿变应原的免疫活性。方法:进行问卷调查及血清特异性IgE(sIgE)检测筛查变应原;采集蒿属样本及内转录间隔区(ITS)序列鉴别物种;构建野艾蒿变应原毕赤酵母蛋白表达载体,转化宿主重组...目的:筛查变应原并明确蒿属植物种属鉴定,深入分析野艾蒿变应原的免疫活性。方法:进行问卷调查及血清特异性IgE(sIgE)检测筛查变应原;采集蒿属样本及内转录间隔区(ITS)序列鉴别物种;构建野艾蒿变应原毕赤酵母蛋白表达载体,转化宿主重组表达和纯化,结果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和western blotting检测分析。结果:376例受试者血清sIgE检测总阳性率为58.78%,其中艾蒿阳性率高达48.67%。ITS序列物种鉴定发现样本与野艾蒿Artemisia lavandulifolia(KX421740)相似性为99.74%;野艾蒿花粉变应原基因Art la 3毕赤酵母分泌表达载体pPICZαA-Art la 3.0101和pPICZαA-Art la 3.0102的片段大小分别为798 bp和795 bp。Western blotting检测结果显示重组变应原Art la 3.0102与16例艾蒿过敏患者血清的IgE结合,阳性率为26.7%(16/60),该16例受试者临床症状较重且艾蒿sIgE等级高。结论:野艾蒿是内蒙古及甘肃地区主要致敏源之一,其重组变应原Art la 3.0102具有IgE结合活性,可作为致敏源筛查的候选抗原,并推测对变应原Art la 3.0102过敏可能作为野艾蒿诱发变应性疾病的病情严重程度指标。展开更多
To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a pro...To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin prote...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.展开更多
文摘目的:筛查变应原并明确蒿属植物种属鉴定,深入分析野艾蒿变应原的免疫活性。方法:进行问卷调查及血清特异性IgE(sIgE)检测筛查变应原;采集蒿属样本及内转录间隔区(ITS)序列鉴别物种;构建野艾蒿变应原毕赤酵母蛋白表达载体,转化宿主重组表达和纯化,结果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和western blotting检测分析。结果:376例受试者血清sIgE检测总阳性率为58.78%,其中艾蒿阳性率高达48.67%。ITS序列物种鉴定发现样本与野艾蒿Artemisia lavandulifolia(KX421740)相似性为99.74%;野艾蒿花粉变应原基因Art la 3毕赤酵母分泌表达载体pPICZαA-Art la 3.0101和pPICZαA-Art la 3.0102的片段大小分别为798 bp和795 bp。Western blotting检测结果显示重组变应原Art la 3.0102与16例艾蒿过敏患者血清的IgE结合,阳性率为26.7%(16/60),该16例受试者临床症状较重且艾蒿sIgE等级高。结论:野艾蒿是内蒙古及甘肃地区主要致敏源之一,其重组变应原Art la 3.0102具有IgE结合活性,可作为致敏源筛查的候选抗原,并推测对变应原Art la 3.0102过敏可能作为野艾蒿诱发变应性疾病的病情严重程度指标。
基金The National Basic Research Program of China(973Program)(No.2011CB933503)the National Natural Science Foundation of China for Key Project of International Cooperation(No.61420106012)China Postdoctoral Science Foundation(No.2013M541592)
文摘To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.
