Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （A...Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （ALV-A） env gene into plasmid pcDNA3.1/Zeo （＋） and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL （50% tissue culture infective dose）ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.展开更多
Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves v...Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.展开更多
Model DF-1 electrochemical analyzer is a many-purpose electrochemical instrumentation with following five functional select switches for operating conditions: cathodic /anodie polarization; single/cycle sean；two/thre...Model DF-1 electrochemical analyzer is a many-purpose electrochemical instrumentation with following five functional select switches for operating conditions: cathodic /anodie polarization; single/cycle sean；two/three electrodes system；direct/derivative output；normal/differential mode. Consequently they can be combined into several working manners, result in different experimental methods as consistent with desire. This instrument has manifold compensator and adjuster with a wide-range, so that the sensitivity and the signal graph have been able to improve. In addition, the instrument incorporates a strip-chart recorder and an exact timer. It is also equipped with electrochemical bench and a set of electrodes including the rotating glassy carbon electrode, gold plate electrode, silver plate electrode, mercury coated silver base electrode, hanging mercury electrode etc. In order to broaden the field of its application, all units of the instrument can be unitized, but each and every can be also used independently. The instrument is applicable to polarography, voltammetry, potentiometric stripping, potentiostatic electrolysis,differential technique, coulometry, linear polarization method, cycle voltammetry, passivation curve, and some other electrochemical technique, which have need for electrochemical cell with two, three，or four electrodes. This paper describes the design fundament, feature and specification of the electrochemical analyzer, and finally gives laboratory application some experimental examples.展开更多
【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利...【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利用qRT-PCR探究不同组织、不同时期circSFMBT2的表达水平。通过构建过表达载体,结合qRT-PCR、Edu和CCK-8等试验,探究circSFMBT2对鸡DF-1细胞增殖的影响。【结果】鸡circSFMBT2全长827 nt,由SFMBT2基因外显子12~18环化形成。RNase R耐受性试验表明,circSFMBT2不易被RNase R降解,随机引物对circSFMBT2的反转录效率是Oligo-d(T)18引物的8倍。组织表达谱表明,circSFMBT2在麒麟鸡14胚龄以及1周龄的肝脏和脾脏中高表达、在胸肌和腿肌中低表达。circSFMBT2在胸肌和腿肌的时序表达谱表明,circSFMBT2在胚胎时期表达量较高,出生后表达量迅速下降。在DF-1细胞中circSFMBT2过表达48 h后,增殖标记基因PCNA和CCND1的表达量分别较对照组上调85%和92%。E d u和C C K-8细胞增殖试验表明,鸡c i r c S F M B T 2可以促进细胞增殖进程。【结论】circSFMBT2是鸡SFMBT2基因的一个环状转录本,可以促进DF-1细胞的增殖,研究结果为深入探究鸡circSFMBT2的生物学功能和作用机制奠定了基础。展开更多
基金The work was founded by the National Key R&D Program of China(2016YFD0501606)the Public Industry Research Program,the Ministry of Agriculture of China(201203055)+2 种基金the Program of Science and Technology Development of Guangdong Province,China(2015A020209145)the China Meat-Type Chicken Research System(CARS-42-G09)the Modern Agriculture Talents Support Program,Ministry of Agriculture of China([2012] no.160)
文摘Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening. As a first step in method development, we inserted the avian leukosis virus subgroup A （ALV-A） env gene into plasmid pcDNA3.1/Zeo （＋） and used this construct to transfect DF-1 cells. Zeocin-resistant cells were obtained after 2 weeks of zeocin selection. Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages. The DF-1/A cell line was completely resistant to 104 TCIDso/0.1 mL （50% tissue culture infective dose）ALV-A and was partially resistant to 10~ TCIDs0/0.1 mL ALV-A viral particles. By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISAfor capsid protein p27. Thus, we established a DF-1/A cell line that was resistant to ALV-A infection. This cell line will be useful as a diagnostic tool.
文摘Modified Vaccinia Ankara(MVA)is a highly promising vector for generating safe vaccine candidates against many pathogens,such as HIV-1,SARS-CoV-2,and influenza viruses.The gold standard method to titrate MVA involves visualizing MVA plaques in chicken embryo fibroblasts after immunostaining.However,this method is time-consuming and costly.In this study,we evaluated the visualization of MVA plaques formed in continuous chicken embryo fibroblasts DF-1 cells using crystal violet staining.We found that MVA titration by plaque assay using crystal violet staining in DF-1 cells yielded similar results to immunostaining,with substantially reduced time and costs.The MVA plaque assay by crystal violet staining in DF-1 cells is a reliable method with accurate results and low time and financial costs.
文摘Model DF-1 electrochemical analyzer is a many-purpose electrochemical instrumentation with following five functional select switches for operating conditions: cathodic /anodie polarization; single/cycle sean；two/three electrodes system；direct/derivative output；normal/differential mode. Consequently they can be combined into several working manners, result in different experimental methods as consistent with desire. This instrument has manifold compensator and adjuster with a wide-range, so that the sensitivity and the signal graph have been able to improve. In addition, the instrument incorporates a strip-chart recorder and an exact timer. It is also equipped with electrochemical bench and a set of electrodes including the rotating glassy carbon electrode, gold plate electrode, silver plate electrode, mercury coated silver base electrode, hanging mercury electrode etc. In order to broaden the field of its application, all units of the instrument can be unitized, but each and every can be also used independently. The instrument is applicable to polarography, voltammetry, potentiometric stripping, potentiostatic electrolysis,differential technique, coulometry, linear polarization method, cycle voltammetry, passivation curve, and some other electrochemical technique, which have need for electrochemical cell with two, three，or four electrodes. This paper describes the design fundament, feature and specification of the electrochemical analyzer, and finally gives laboratory application some experimental examples.
文摘【目的】验证鸡circSFMBT2的环形结构,探究其表达规律及功能。【方法】以麒麟鸡和DF-1细胞为研究对象,根据反向剪切位点序列特征设计引物,通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性,利用qRT-PCR探究不同组织、不同时期circSFMBT2的表达水平。通过构建过表达载体,结合qRT-PCR、Edu和CCK-8等试验,探究circSFMBT2对鸡DF-1细胞增殖的影响。【结果】鸡circSFMBT2全长827 nt,由SFMBT2基因外显子12~18环化形成。RNase R耐受性试验表明,circSFMBT2不易被RNase R降解,随机引物对circSFMBT2的反转录效率是Oligo-d(T)18引物的8倍。组织表达谱表明,circSFMBT2在麒麟鸡14胚龄以及1周龄的肝脏和脾脏中高表达、在胸肌和腿肌中低表达。circSFMBT2在胸肌和腿肌的时序表达谱表明,circSFMBT2在胚胎时期表达量较高,出生后表达量迅速下降。在DF-1细胞中circSFMBT2过表达48 h后,增殖标记基因PCNA和CCND1的表达量分别较对照组上调85%和92%。E d u和C C K-8细胞增殖试验表明,鸡c i r c S F M B T 2可以促进细胞增殖进程。【结论】circSFMBT2是鸡SFMBT2基因的一个环状转录本,可以促进DF-1细胞的增殖,研究结果为深入探究鸡circSFMBT2的生物学功能和作用机制奠定了基础。
