| 猪繁殖与呼吸综合征病毒糖基化囊膜蛋白5原核表达载体的构建及免疫原性分析 |
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| 引用本文： | 李欣贤,张晓丹,刘晶晶,苗增民,张涛涛,柴同杰.猪繁殖与呼吸综合征病毒糖基化囊膜蛋白5原核表达载体的构建及免疫原性分析[J].中国畜牧兽医,2014,41(4):89-94. |
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| 作者姓名： | 李欣贤 张晓丹 刘晶晶 苗增民 张涛涛 柴同杰 |
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| 作者单位： | (1.山东农业大学动物科技与动物医学学院，山东泰安 271000；2.泰山医学院生命科学学院，山东泰安 271000；3.石家庄兽用生物制品供应站，河北石家庄 050000) |
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| 基金项目： | 国家科技支撑计划（2012BAD39B02）。 |
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| 摘 要： | 为分析猪繁殖与呼吸综合征病毒(PRRSV)糖基化囊膜蛋白5（GP5）的免疫原性，本研究通过提取PRRSV分离株（GenBank登录号：HQ701732.1）RNA和RT-PCR扩增得到开放阅读框5（ORF5）基因。根据ORF5的基因序列，设计2对引物，经PCR扩增分别获得不含信号肽和跨膜功能区的2段基因片段。利用酶切位点，将2段基因连接到原核表达载体pET-28a（+）上，获得重组表达质粒pET28a-GP5。将重组质粒导入BL21（DE3）感受态细胞，经IPTG诱导获得表达。经Western blotting鉴定，重组蛋白可被PRRSV阳性血清识别。将纯化的重组蛋白免疫BALB/c小鼠，ELISA方法检测，小鼠能产生针对蛋白的血清抗体。因此，该重组PRRSV GP5蛋白具有良好的生物学活性，为进一步研究GP5蛋白的结构和功能奠定基础。
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| 收稿时间： | 2013-11-07 |
Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus |
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| LI Xin-xian,ZHANG Xiao-dan,LIU Jing-jing,MIAO Zeng-min,ZHANG Tao-tao,CHAI Tong-jie.Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus[J].China Animal Husbandry & Veterinary Medicine,2014,41(4):89-94. |
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| Authors: | LI Xin-xian ZHANG Xiao-dan LIU Jing-jing MIAO Zeng-min ZHANG Tao-tao CHAI Tong-jie |
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| Institution: | (1.College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an 271000, China; 2. College of Life Science, Taishan Medical University, Tai’an 271000, China;3.Veterinary Biologics Supply Station of Shijiazhuang, Shijiazhuang 050000, China) |
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| Abstract: | In order to analyze the immunogenicity of glycosylated envelope protein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV), ORF5 gene fragment was amplified by RT-PCR from the PRRSV (GenBank: HQ701732.1). Based on the ORF5 gene sequence, two pairs of primers were used to amplify two gene fragments, excluding the signal peptide sequence and transmembrane regions. The two gene fragments were cloned into the prokaryotic expression vector pET-28a（+）, and then the recombinant plasmid was transformed into Escherichia coli. The results of Western blotting and ELISA respectively showed that the expression of GP5 could be recognized by positive serum antibody of PRRSV, and corresponding antibodies of GP5 protein could produce in the immunized BALB/c mice. Therefore, the recombinant GP5 protein had good biological activity, and could provide fundamental data for the further study on the structure and function of GP5 protein of PRRSV. |
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| Keywords: | porcine reproductive and respiratory syndrome virus glycosylated envelope protein 5 recombinant protein ELISA Western blotting |
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