埃博拉病毒GP和VP40蛋白在哺乳动物细胞中的共表达及病毒样颗粒装配

目的：制备基因重组埃博拉病毒样颗粒，为疫苗研究及埃博拉病毒特异抗原、抗体检测提供基础。方法：根据埃博拉病毒扎伊尔株的GP和VP40蛋白氨基酸序列，以哺乳动物细胞基因表达密码子偏好性进行基因优化设计；化学合成GP和VP40基因片段并分别构建于表达质粒pcDNA3.1或同时构建到具有双表达单元的质粒pBudCE4.1；重组质粒经lipofectamine2000转染293FT细胞；以Western blot检测重组蛋白GP和VP40的表达；通过电镜观察病毒样颗粒。结果：构建的重组质粒经酶切鉴定及测序分析证实构建成功；Western blot结果显示，共转染分别表达GP和VP40的两个质粒或转染共表达两个蛋白的质粒都发现GP特异反应条带产生，且大小与预期相符，此外，转染共表达质粒产生的GP蛋白表达明显强于两个质粒共转染，并同时可检测到VP40的表达；电镜观察到典型的丝状的埃博拉病毒样颗粒。结论：在293FT细胞中基因优化的埃博拉病毒GP和VP40可有效表达并装配为病毒样颗粒，为进一步研究奠定了基础。

Co-expression of Ebola Virus GP and VP40 Proteins and Virus-like particles Assembly in Mammalian Cells

Objective: To co-expression Ebola virus proteins GP and VP40 in mammalian cells and generate Ebola virus-like particles. Methods: According to amino acid sequences, the nucleotide sequences of Ebola-Zaire GP and VP40 genes were optimized based on the codon usage bias in mammalian cells. The synthesized genes were cloned into expression plasmids pcDNA3.1 or pBudCE4.1 which has two expression units, and then 293FT cells were transfected with the recombinant plasmids using lipofectamine2000. The expression of recombinant proteins was detected by Western blot, and the VLPs were observed by electron microscope. Results: Specific reactive bands recognized by anti-GP antibody were found in cells transfected with the recombinant plasmid carrying optimized GP gene. The expression of recombinant GP protein mediated by the plasmid pBudCE4.1/GP/VP40 which co-expressing GP and VP40 was significantly higher than that mediated by co-transfection with the plasmids pcDNA3.1/GP which expresses GP and pcDNA3.1/VP40 which expresses VP40. Besides, VP40 expression was detectable although the level was low. Classical Ebola virus-like particles were found under the observation with electron microscope. Conclusion: Ebola virus proteins GP and VP40 were successfully expressed and assembled into virus-like particles in 293FT cells, which laid an important foundation for further studies on the development of a vaccine or virus detection reagent.