重组中东呼吸综合征病毒刺突蛋白S1亚基及其编码DNA接种小鼠的抗血清水平研究

doi:10.13560/j.cnki.biotech.bull.1985.2018-0273

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 150-156.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0273

重组中东呼吸综合征病毒刺突蛋白S1亚基及其编码DNA接种小鼠的抗血清水平研究

张倩倩^{1, 2}, 刘康泰², 韩宗晟², 李荣贵¹

1. 青岛大学生命科学学院,青岛 266071;
2. 中国医学科学院基础医学研究所,北京 100005

收稿日期:2018-03-27 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:张倩倩,女,硕士研究生,研究方向：生物工程;E-mail：alicezhangqian@yeah.net
基金资助:
中国医学科学院医学与健康科技创新工程（2017-12M-1-009）

Antiserum Levels in Mice Inoculated with Spike Protein S1 Subunit and Its Coding DNA from Recombinant Middle East Respiratory Syndrome Virus

ZHANG Qian-qian^{1, 2}, LIU Kang-tai², HAN Zong-sheng², LI Rong-gui¹

1. College of Life Sciences,Qingdao University,Qingdao 266071;
2. Institute of Basic Medical Science,Chinese Academy of Medical Science,Beijing 100005

Received:2018-03-27 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

摘要： 人工合成了大小为2 253 bp刺突蛋白的S1亚基的基因片段,构建了含有中东呼吸综合征冠状病毒刺突蛋白S1亚单位（MERS-Co V S1）编码基因的重组表达质粒Peak13CD5LS1TEVhIgGFc,转染重组质粒至HEK293E细胞后,筛选出了可稳定表达重组融合蛋白的阳性克隆细胞株,进行了刺突蛋白亚单位融合蛋白（S1-Fc）的重组表达和纯化,获得了大小约为122 kD的重组S1-Fc融合蛋白,Western blotting验证重组蛋白为目的蛋白;将重组质粒Peak13CD5LS1TEVhIgGFc、重组S1-Fc融合蛋白以及Peak13CD5LS1TEVhIgGFc联合S1-Fc融合蛋白分别接种小鼠,以纯化的重组S1-Fc融合蛋白作为抗原,利用Western blotting及ELISA分别检测了小鼠的抗S1血清滴度及特异性。结果发现,3种方法接种的小鼠中均存在特异性抗S1血清。其中,接种重组S1-Fc融合蛋白和接种S1-Fc融合蛋白联合重组质粒的小鼠在首次接种就产生高滴度抗S1血清,而只接种重组质粒的小鼠在3次接种后才产生了特异性抗S1血清。两种含重组S1-Fc融合蛋白的接种方法诱导小鼠产生的抗S1血清滴度基本一致,均显著高于仅接种重组质粒的小鼠的抗S1血清,证明短期内重组S1-Fc蛋白接种更快速高效。

关键词: 中东呼吸综合征冠状病毒, 重组质粒, 重组S1-Fc, 接种, 血清滴度

Abstract: Gene fragment of S1 subunit of spike protein was artificially synthesized. Then recombinant expression plasmid Peak13CD5LTEVhIgGFc containing MERS（Middle East respiratory syndrome）coronavirus spike protein（MERS-Co V S1）was constructed and transfected into HEK293E cells,and a positive monoclonal cell line of that may stably express recombinant fusion protein was selected. The recombinant fusion protein S1-Fc with a molecular weight of 122 kD was obtained after expression and purification,and confirmed as the target protein by Western blotting. Mice were inoculated with the recombinant plasmid Peak13CD5LS1TEVhIgGFc,the recombinant S1-Fc protein,and the combination of the recombinant plasmid and S1-Fc protein,respectively. The purified recombinant S1-Fc was used as the antigen,and the titer and specificity of the antiserum of the test mice were detected by Western blotting and ELISA. The results showed that specific S1 antiserums were all present in the mice immunized with the three different inoculations. Among them,the mice immunized with the recombinant S1-Fc protein and the combination of recombinant S1-Fc protein and Peak13CD5LS1TEVhIgGFc produced high titers of antiserums against S1 after the first time of the immunization,while the mice immunized only by recombinant plasmid produced antiserum after 3 times immunizations. The antiserums prepared by immunization with the recombinant S1-Fc and the combination of recombinant S1-Fc and recombinant plasmid had the consistent antiserum titer,higher than those in the mice immunized only with the recombinant plasmid,suggesting that the inoculation of recombinant S1-Fc protein in short term was fast and efficient.

Key words: Middle East respiratory syndrome coronavirus, recombinant plasmid, recombinant S1-Fc, inoculation, serum titer

张倩倩, 刘康泰, 韩宗晟, 李荣贵. 重组中东呼吸综合征病毒刺突蛋白S1亚基及其编码DNA接种小鼠的抗血清水平研究[J]. 生物技术通报, 2018, 34(10): 150-156.

ZHANG Qian-qian, LIU Kang-tai, HAN Zong-sheng, LI Rong-gui. Antiserum Levels in Mice Inoculated with Spike Protein S1 Subunit and Its Coding DNA from Recombinant Middle East Respiratory Syndrome Virus[J]. Biotechnology Bulletin, 2018, 34(10): 150-156.

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重组中东呼吸综合征病毒刺突蛋白S1亚基及其编码DNA接种小鼠的抗血清水平研究

Antiserum Levels in Mice Inoculated with Spike Protein S1 Subunit and Its Coding DNA from Recombinant Middle East Respiratory Syndrome Virus

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