狂犬病病毒受体P75NTR原核表达的抗原性分析及多克隆抗体制备
详细信息 查看官网全文
- 英文论文题名：Study on antigenicity of rabies virus neurotrophin receptor P75 by prokaryotic expression and preparation of polyclonal antibody
- 论文作者：钟桃珍 ; 邢杏伟 ; 廖芳 ; 彭可可 ; 邓朝谦 ; 蒋元 ; 欧阳伶宣 ; 覃雨阳 ; 廖素环 ; 梁晶晶 ; 罗廷荣
- 英文论文作者：Taozhen Zhong ; Xingwei Xing ; Fang Liao ; Keke Peng ; Chaoqian Deng ; Yuan Jiang ; Lingxuan Ouyang ; Ting Rong Luo ; State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresourses ; Guangxi University ; Laboratory of Animal Infectious Diseases ; College of Animal Sciences and Veterinary Medicine ; Guangxi University
- 年：2016
- 作者机构：广西大学动物科学技术学院动物传染病实验室;广西大学亚热带农业生物资源保护利用国家重点实验室;
- 论文关键词：狂犬病病毒 ; 神经营养因子受体P75(P75NTR) ; 抗原性 ; 原核表达 ; 多克隆抗体
- 英文论文关键词：Rabies virus(RV) ; Neurotrophin receptor P75(P75NTR) ; antigenicity ; prokaryotic expression ; polyclonal antibody
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.65
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：8
- 文件大小：726k
- 原文格式：D
- 会议级别：全国
摘要
【目的】研究狂犬病病毒(RV)神经营养因子受体P75(P75NTR)原核表达的抗原性及制备出多克隆抗体,为进一步研究RV的致病机制奠定基础。【方法】采用RT-PCR从感染Flury株的NA细胞中克隆P75NTR基因,选取P75NTR基因的418~681 nt和831~1080 nt的两个区域共513 nt编码171个氨基酸的区域,与p ET-32a(+)重组构建原核表达载体,然后转化BL-21(DE3)感受态细胞进行表达,再以纯化的重组蛋白P75NTR免疫昆明小鼠制备本狂犬病病毒受体P75NTR抗体,并以Western blotting和间接免疫荧光技术(IFA)检测其抗原性。【结果】以原核表达载体p ET-P75-513转化BL21(DE3)感受态细胞,经0.2 mmol/L IPTG诱导6 h可获得较高表达量的P75NTR重组蛋白,且主要以可溶性蛋白形式进行表达,可用Ni-NTA树脂分离柱进行纯化。经Western blotting和IFA检测,发现制备获得的抗P75NTR多抗能与NA细胞表面自然的P75NTR及转染BHK-21细胞表达的P75NTR发生良好反应,且抗体效价高,可达1:16000。【结论】
【Objective】 The neurotrophin receptor P75(P75NTR) as a receptor of rabies virus(RV) binds with the RV glycoprotein and correlates RV virion into cytoplasm for replication.The anti-P75 NTR polyclonal antibody will help to explore the interactive mechanism between RV and P75 NTR.【Method】In this study,the total RNA were extracted from NA cells inoculated with RV Flury strain for 1 hour and the P75 NTR gene was amplified by RT-PCR.The PCR products were used to construct a recombinant prokaryotic expression plasmid p ET-P75-513 containing a total of 513 nucleotides(nt)containing 418-681 nt and 831-1080 nt of P75 NTR,and the plasmid p ET-P75-513 was transformed into competent cell E.coli BL21 for expression.【Results】The results showed that the expressed-product fusing 171 amino acidsof P75 NTR and thioredoxin was confirmed as a soluble form and was purified by nickel-ion affinity chromatography.The purified P75 NTR was used to immune the Kunming mice for preparation of anti-P75 NTR polyclonal antibody.【Conclusion】Indirect fluresence assay(IFA) and western blot analysis showed that the anti-P75 NTRpolyclonal antibody could well react with the natural P75 NTR on the surface of NA cells and the expressed P75 NTR on thesurface of transfected BHK cell withhigh specific and high titers.These results indicated that the prokaryotic expressed P75 NTR had well antigenicity.
【Objective】 The neurotrophin receptor P75(P75NTR) as a receptor of rabies virus(RV) binds with the RV glycoprotein and correlates RV virion into cytoplasm for replication.The anti-P75 NTR polyclonal antibody will help to explore the interactive mechanism between RV and P75 NTR.【Method】In this study,the total RNA were extracted from NA cells inoculated with RV Flury strain for 1 hour and the P75 NTR gene was amplified by RT-PCR.The PCR products were used to construct a recombinant prokaryotic expression plasmid p ET-P75-513 containing a total of 513 nucleotides(nt)containing 418-681 nt and 831-1080 nt of P75 NTR,and the plasmid p ET-P75-513 was transformed into competent cell E.coli BL21 for expression.【Results】The results showed that the expressed-product fusing 171 amino acidsof P75 NTR and thioredoxin was confirmed as a soluble form and was purified by nickel-ion affinity chromatography.The purified P75 NTR was used to immune the Kunming mice for preparation of anti-P75 NTR polyclonal antibody.【Conclusion】Indirect fluresence assay(IFA) and western blot analysis showed that the anti-P75 NTRpolyclonal antibody could well react with the natural P75 NTR on the surface of NA cells and the expressed P75 NTR on thesurface of transfected BHK cell withhigh specific and high titers.These results indicated that the prokaryotic expressed P75 NTR had well antigenicity.
引文