裂谷热病毒Gn蛋白主要抗原区的串联表达和多克隆抗体制备
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“十三五”国家重点研发计划项目(2016YFD0500908)


Tandem Expression of the Major Antigenic Areas of RVFV Gn Protein and the Preparation of Polyclonal Antibodies
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    摘要:

    裂谷热(Rift Valley fever,RVF)是由裂谷热病毒(Rift Valley fever virus,RVFV)引起的一种烈性人兽共患传染病。RVFV囊膜蛋白Gn可诱导产生中和抗体,是RVFV检测方法和疫苗研究的重要抗原靶标。本研究通过分析蛋白抗原位点信息,构建包含Gn蛋白两个主要抗原区域的重组表达载体,随后将质粒转化至BL21感受态细胞,以IPTG诱导重组蛋白表达并优化蛋白表达条件,通过Western Blot鉴定重组蛋白;将重组蛋白免疫BALB/c小鼠,制备多克隆抗体,并以ELISA、Western Blot、IFA检测多克隆抗体的反应性。结果显示:诱导表达的Gn重组蛋白分子质量约为45 kDa;蛋白表达条件优化为IPTG终浓度0.25 mmol/L,诱导时间5 h;Western Blot鉴定发现蛋白成功表达。通过ELISA测定小鼠三免后血清抗体,结果发现抗体效价大于1:51 200;Western Blot检测显示,制备的多抗血清能与重组蛋白发生反应;进一步的IFA检测结果表明,制备的多克隆抗体可与真核质粒转染细胞中表达的Gn蛋白反应。本研究获得的Gn重组蛋白及制备的多克隆抗体为后续RVFV检测方法的建立奠定了基础。

    Abstract:

    Rift Valley fever(RVF)is a virulent zoonotic disease caused by Rift Valley fever virus(RVFV). The envelope protein Gn of RVFV is an important antigen target for the detection method development and vaccine research,as it could induce the production of neutralizing antibodies. In the study,the recombinant expression vector containing two major antigen areas of Gn protein was constructed through analyzing the information of protein antigen sites,then the plasmid was transformed into BL21 cells,the recombinant protein expression was induced by IPTG,followed by optimization of relevant conditions,the recombinant protein was identified by Western Blot and then vaccinated into BALB/c mice to prepare the polyclonal antibodies that were tested by ELISA,Western Blot and IFA. The results showed that the molecular weight of the induced Gn recombinant protein was about 45 kDa;the expression conditions were optimized as follows:the final concentration of IPTG was 0.25 mmol/L with the induction time of 5 h;and the protein was successfully expressed as identified by Western Blot. The antibody titer in vaccinated mice was higher than 1:51 200 as tested by ELISA;it was shown by Western Blot that,the prepared polyclonal antiserum could react with the recombinant protein;and the polyclonal antibodies could react with the Gn protein expressed in eukaryotic transfection cells as detected by IFA. In conclusion,future development of RVF detection methods was provided with a basis by the expression of Gn protein and preparation of polyclonal antibodies.

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恽佳蕾,李文良,毛立,杨蕾蕾,孙敏,张纹纹,刘茂军,.裂谷热病毒Gn蛋白主要抗原区的串联表达和多克隆抗体制备[J].《中国动物检疫》编辑部,2021,38(2):108-113.

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  • 在线发布日期: 2021-02-01
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