中国媒介生物学及控制杂志

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汉坦病毒S基因在原核细胞中高效表达的研究

袁子国1,2;张秀香1;张守峰1; 徐慧娟1; 王晓虎1; 扈荣良1   

  1. 1军事医学科学院军事兽医研究所 长春130062;2华南农业大学兽医学院 广州510642
  • 出版日期:2008-06-20 发布日期:2008-06-20

Study on high-level expression of Hantavirus S gene in prokaryocyte

YUAN Zi-guo1;2; ZHANG Xiu-xiang; ZHANG Shou-feng; XU Hui-juan; WANG Xiao-hu; HU Rong-liang   

  1. 1 Veterinary Institute, Academy of Military Medical Science, Changchun 130062, China; 2 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
  • Online:2008-06-20 Published:2008-06-20

摘要: 目的 通过基因工程的方法从鼠肺中扩增出汉坦病毒的S基因,并实现其蛋白的体外表达。方法 应用RT-PCR方法扩增汉坦病毒汉城型(SEO)的YZG-Changchun株S基因,然后克隆到pMD18-T载体中,经序列分析后,定向克隆入原核表达载体pET-28a中,转化大肠埃希菌Rosetta用IPTG诱导表达后,将全菌裂解,用SDS-PAGE和Western-blot检测重组菌中外源蛋白的表达情况和免疫反应性的分析。结果 S基因在原核细胞中得到了高效表达,表达的蛋白占菌体蛋白总量的37%,且具有良好的免疫反应性。结论 汉坦病毒S基因蛋白可通过基因工程手段获得体外高效表达,这将对其功能的研究及为汉坦病毒疫苗的研究提供基础。

关键词: 汉坦病毒, S基因, 原核细胞, 表达

Abstract: Objective To clone S gene from the lung of mice and to realize the expression of its protein in vitro. Methods The S genes of YZG-Changchun stains of Hantavirus were amplified by RT-PCR, and then the RT-PCR products were cloned into the pMD18-T vector. After sequencing analysis, the foreign gene in the recombinant plasmid was cut by restriction enzymes and cloned into the expression vector pET-28a. The recombinant plasmid was then transformed into E.coli Rosetta induced with IPTG. To detect the expression and the immunoreactive of exogenous protein in recombination plasmid by SDS-PAGE and Western-blot. Results The fusion protein was about 37% of the total proteins by gel thin scanner instrument CS-9000 and possessed favourable immunoreactive. Conclusion The gene of S protein can be expressed in a high efficiency in vitro by genetic engineering method, so it provides a good basis for further research on its function and the development of Hantavirus vaccine.