国家科技支撑计划(2011BAI15B03);十二五重大传染病专项(2011ZX09201-031)。
目的 获得猕猴B病毒gB蛋白胞外区基因,并分析其表达特性。方法 利用基因合成的方法合成B病毒gB蛋白胞外区的基因片段,经BamHⅠ和NotⅠ双酶切后连接到pEGFP-N3载体。将构建的pEGFP-N3-GB合重组质粒转染到293细胞。提取蛋白用Western blot检测其在细胞内的表达情况,并利用激光共聚焦分析在细胞内的表达定位。 结果 pEGFP-N3-GB合重组质粒能在293细胞内正常表达, 并且在细胞膜上表达。 结论 利用真核表达系统,可以产生B病毒gB蛋白的特异性抗原重组蛋白,且在细胞膜上表达。
Objective To get the gene encoding extracellular domains of gB protein of B virus and analyze its expression in the eukaryocyte cell. Methods synthesizing gene fragment encoding extracellular domains of gB protein of B virus was by using synthesis gene, then digested with the restriction endonucleases BamHⅠand NotⅠand inserted into eukaryotic expressing vector pEGFP-N3. pEGFP-N3-GB合 was transfected into 293 cells. After protein extraction, the expression of gene was detcted by western blotting, and the cellular localization of the gene was analyzed by immunofluorescence and laser scanning confocal microscopy. Results pEGFP-N3-GB合 were expressed in 293 cells and on the cell membrane. Conclusion eukaryotic expressing system can produce specific antigen recombination protein of B virus gB protein and express on the cell membrane.
刘慧芳,孙书芳,曾林,易思萌,游颖,马雨楠,范君文,孙兆增,王新.猕猴B病毒gB蛋白胞外区基因片段的合成和真核表达[J].中国比较医学杂志,2014,24(11):6~9.
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