应用2A肽策略构建双基因共表达转基因斑马鱼的研究

The Study on Application of Bicistronic Vector in Transgenic Zebrafish Based on 2A Peptide

目的以Tol2为骨架载体,以绿色荧光蛋白(GFP)、Cherry为报告基因,探讨采用2A肽双基因载体构建策略构建单启动子双基因共表达质粒的方法;将B细胞刺激因子(BAFF)分别置于2A序列前后位置,分析位置效应对跨膜融合蛋白的表达与剪切的影响,探讨多基因共表达转基因斑马鱼构建技术。方法以In Fusion法将GFP-2A-Cherry序列构建到Tol2质粒上,所得p Tol-GFP-2A-Cherry质粒转染He La细胞、显微注射1-细胞期斑马鱼受精卵;倒置荧光显微镜观察He La细胞、斑马鱼幼鱼体内GFP与Cherry蛋白的表达,Western blot法验证GFP和Cherry蛋白的表达量与剪切情况;分别构建p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry质粒,Western blot法检查BAFF的表达与剪切情况。结果 p Tol2-GFP-2A-Cherry质粒转染的He La细胞,GFP与Cherry均可单独表达且表达呈现时空一致性;GFP-2A-Cherry融合蛋白可被剪切为GFP与Cherry,且成等比例表达趋势。p Tol2-GFP-2A-Cherry质粒显微注射1-细胞期斑马鱼受精卵可获得可单独表达GFP与Cherry蛋白的转基因斑马鱼;p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry于斑马鱼体内均有融合蛋白的表达,且BAFF序列位于2A序列后更易于融合蛋白的剪切。结论通过2A肽策略构建可实现在斑马鱼体内单一载体、单一启动子调控双基因表达目的。发现编码跨膜分泌蛋白的功能基因位于2A序列的不同位置会直接影响蛋白的剪切,功能基因位于2A序列后易于跨膜蛋白的剪切。

Objective To apply 2A peptide in generating dicistronic or polycistronic transgenic zebrafish. Methods GFP and Cherry were constructed to the To12 plasmid backbone ; B cell stimulating factor （BAFF） was also inserted into the upstream or downstream of 2A sequence to analysis the position impact on transmembrane fusion protein expression, and to explore the multi-gene co-expression of transgenic zebrafish. GFP-2A-Cherry sequence was cloned into the To12 plasmid by InFusion method to obtain pTol-GFP-2A-Cherry plasmid, and this plasmid was then transfected into HeLa cells and microin- jected into 1-cell stage fertilized embryos of zebrafish; fluorescence microscopy was used to trace the GFP and Cherry ex- pression of HeLa cells in vitro and juvenile in vivo. the expression of GFP and Cherry protein was validated by Western blot ; BAFF, as a candidate gene, was constructed as pTol2-GFP-2A-BAFF and pTo12-BAFF-2A-Cherry, and were then injected into 1-cell stage fertilized embryos followed by Western blot to determine the 2A＇ s position effects on protein expression. Results GFP and Cherry would individually express and showed consistently temporal expression; GFP-2A-Cherry fusion protein could be spliced into GFP and Cherry proportionally. The pTo12-GFP-2A-Cherry plasmid which was microinjected into 1-cell stage fertilized embryos could express GFP and Cherry protein alone; Both pToI2-GFP-2A-BAFF and pTol2- BAFF-2A-Cherry could express fusion protein in zebrafish, however, BAFF that located downstream of 2A peptide was easi- ly cleavaged from the fusion protein. Conclusion 2A peptide building strategy can be carried out in zebrafish with single carrier and single promoters, therefore, it was proved that the use of 2A peptide strategy could achieve muhi-gene expres- sion. And it was also found that the localization of 2A peptide could directly affect the protein function.