| 非洲猪瘟病毒外膜蛋白CD2v的原核表达及其免疫特性的研究 |
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| 引用本文： | 钟秋萍,于婉琪,薄宗义,吕璐,谢振华,王振忠,郑龙三,张志芳,扈荣良,钱莺娟,陈鸿军.非洲猪瘟病毒外膜蛋白CD2v的原核表达及其免疫特性的研究[J].中国兽医科学,2020(6):689-695. |
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| 作者姓名： | 钟秋萍 于婉琪 薄宗义 吕璐 谢振华 王振忠 郑龙三 张志芳 扈荣良 钱莺娟 陈鸿军 |
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| 作者单位： | 南京农业大学动物医学院;中国农业科学院上海兽医研究所;中国人民解放军军事医学科学院军事兽医研究所;中国农业科学院生物技术研究所 |
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| 基金项目： | “十三五”国家重点研发专项资助项目(2017YFD0502300,2018YFC08400400);国家自然科学基金联合基金项目重点支持项目—区域创新发展联合基金项目(U19A2039)。 |
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| 摘 要： | 本研究参考非洲猪瘟病毒(ASFV)国内分离株SY18株的EP402R基因(编码CD2v蛋白),优化其序列,设计特异性引物扩增。将去除跨膜保留胞外区或胞内区的两种截短体克隆到原核表达载体p ET-28a中,再转化至大肠杆菌BL21(DE3)感受态细胞。经IPTG诱导,铁蛋白融合CD2v胞外区(CD2vN-Fe)和铁蛋白融合CD2v胞外-胞内区(CD2v-NC-Fe)均获得高效表达。以该融合蛋白为抗原免疫BALB/c小鼠制备多克隆抗体,通过酶联免疫吸附实验(ELISA)和间接免疫荧光(IFA)方法检测其灵敏度和特异性。结果表明:CD2vN-Fe多抗特异性较强。本研究为建立ASFV的血清学鉴别检测方法奠定了基础。
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| 关 键 词： | 非洲猪瘟病毒 CD2v 原核表达 抗体 |
Prokaryotic expression and immunological characterization of African swine fever virus out membrane CD2v protein |
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| ZHONG Qiu-ping,YU Wan-qi,BO Zong-yi,LV LU,XIE Zhen-hua,WANG Zhen-zhong,YONG-Sam Jung,ZHANG Zhi-fang,HU Rong-liang,QIAN Ying-juan,CHEN Hong-jun.Prokaryotic expression and immunological characterization of African swine fever virus out membrane CD2v protein[J].Veterinary Science in China,2020(6):689-695. |
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| Authors: | ZHONG Qiu-ping YU Wan-qi BO Zong-yi LV LU XIE Zhen-hua WANG Zhen-zhong YONG-Sam Jung ZHANG Zhi-fang HU Rong-liang QIAN Ying-juan CHEN Hong-jun |
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| Institution: | (College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Institute of Military Veterinary Medicine,PLA Academy of Military Medical Sciences,Changchun 130122,China;Biotechnology Research Institute,CAAS,Beijing 100081,China) |
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| Abstract: | In this study,the sequence of EP402 R gene encoding CD2 v from African swine fever virus(ASFV)Chinese strain SY18 was optimized and synthesized.The extracellular domain of CD2 v fused with ferritin(CD2 v N-Fe)and the extracellular-intracellular domain of CD2 v fused with ferritin(CD2 v-NC-Fe)were amplified by fusion PCR.Different fragments were subcloned into prokaryotic expression vector p ET-28 a and transformed into E.coli BL21(DE3)competent cells.Induced by Isopropylβ-D-thiogalactoside(IPTG),CD2 v-NC-Fe and CD2 v N-Fe fragments were highly expressed.The fusion proteins were used as antigen to immunize Balb/c mice to prepare antibodies.The sensitivity and specificity of antisera were detected by ELISA and IFA.The results showed that the specificity of antibodies against CD2 v N-Fe protein was strong,which laid a foundation for the establishment of a serological method for the detection of ASFV. |
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| Keywords: | African swine fever virus CD2v prokaryotic expression antibody |
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