旨在构建ALV-J受体分子chNHE1精准基因编辑细胞系,本研究利用荧光标记的CRISPR/Cas9系统,在DF-1细胞中将chNHE1介导ALV-J进入宿主细胞的关键氨基酸V33进行突变,W38进行缺失,同时将编码第34-37位氨基酸的密码子同义替换。通过流式细胞分...旨在构建ALV-J受体分子chNHE1精准基因编辑细胞系,本研究利用荧光标记的CRISPR/Cas9系统,在DF-1细胞中将chNHE1介导ALV-J进入宿主细胞的关键氨基酸V33进行突变,W38进行缺失,同时将编码第34-37位氨基酸的密码子同义替换。通过流式细胞分选获得48株单克隆细胞系,PCR及测序分析结果显示,其中有14株单克隆细胞系的chNHE1成功发生V33突变、W38缺失以及34-37位氨基酸的密码子同义替换,基因编辑效率为29%。为了验证chNHE1基因编辑DF-1细胞系的遗传稳定性及增殖水平,对传至第25代的细胞系进行测序分析,结果显示,chNHE1基因未发生回复性突变;进一步细胞计数分析结果显示,chNHE1基因编辑细胞系增殖水平未受到影响;为了评价chNHE1基因编辑细胞系抗ALV-J感染的能力,分别利用ALV-J荧光报告病毒(ALV-J-GFP)及ALV-J原型毒株(HPRS-103)对其进行病毒感染试验,荧光观察结果及流式细胞分析结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI ALV-J-GFP的感染;进一步间接免疫荧光试验、PCR扩增试验以及病毒滴度测定试验结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI HPRS-103毒株及0.1 MOI JL08CH3-1毒株的感染。本研究利用荧光标记的CRISPR/Cas9系统结合流式细胞分选,成功构建了chNHE1基因编辑细胞系,其可完全抵抗ALV-J的感染,且该细胞系遗传稳定性及增殖活性良好,为建立抗ALV-J感染的新技术提供了理论支持及基因编辑靶点。展开更多
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
文摘旨在构建ALV-J受体分子chNHE1精准基因编辑细胞系,本研究利用荧光标记的CRISPR/Cas9系统,在DF-1细胞中将chNHE1介导ALV-J进入宿主细胞的关键氨基酸V33进行突变,W38进行缺失,同时将编码第34-37位氨基酸的密码子同义替换。通过流式细胞分选获得48株单克隆细胞系,PCR及测序分析结果显示,其中有14株单克隆细胞系的chNHE1成功发生V33突变、W38缺失以及34-37位氨基酸的密码子同义替换,基因编辑效率为29%。为了验证chNHE1基因编辑DF-1细胞系的遗传稳定性及增殖水平,对传至第25代的细胞系进行测序分析,结果显示,chNHE1基因未发生回复性突变;进一步细胞计数分析结果显示,chNHE1基因编辑细胞系增殖水平未受到影响;为了评价chNHE1基因编辑细胞系抗ALV-J感染的能力,分别利用ALV-J荧光报告病毒(ALV-J-GFP)及ALV-J原型毒株(HPRS-103)对其进行病毒感染试验,荧光观察结果及流式细胞分析结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI ALV-J-GFP的感染;进一步间接免疫荧光试验、PCR扩增试验以及病毒滴度测定试验结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI HPRS-103毒株及0.1 MOI JL08CH3-1毒株的感染。本研究利用荧光标记的CRISPR/Cas9系统结合流式细胞分选,成功构建了chNHE1基因编辑细胞系,其可完全抵抗ALV-J的感染,且该细胞系遗传稳定性及增殖活性良好,为建立抗ALV-J感染的新技术提供了理论支持及基因编辑靶点。
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.
