植物蛋白融合HA标签通用载体构建与应用

以植物表达载体pCamE(GenBank No.:JX841315)为基本骨架,HA蛋白YPYDVPDYA为标签序列,构建可分别融合目标蛋白N端与C端表达载体pCHAN与pCHAC,并增加有利外源基因插入的酶切位点,使之成为含有7个常见限制性内切酶(PstⅠ、SpeⅠ、XbaⅠ、Bam HⅠ、KpnⅠ、SmaⅠ、ApaⅠ或SalⅠ)单一识别位点的通用型标签载体;为验证该载体实用性,将项目组自主克隆的大豆紫色酸性磷酸酶GmPAP14(GenBank No.:JN967626)连接至上述载体,并转入拟南芥;经PCR检测与测序分析获得T2转基因株系,HA标签抗体Western blotting结果发现转pCHAN-GmPAP14、pCHAC-GmPAP14拟南芥可获得目的蛋白融合HA标签杂交条带,而对照无该条带出现,证明2个HA标签载体能够稳定融合外源基因编码蛋白N端与C端,且可在转基因植物中正常翻译表达,为植物蛋白分离纯化及相关领域研究提供了稳定可靠的通用型融合标签载体资源。

Construction and Amplification of Protein Fusion HA Tag Universal Expression Vector in Plant

With plant expression vector pCamE (GenBank No.:JX841315) as basic backbone, and HA protein YPYDVPDYA as tag sequence, two protein N and C terminal fusion HA tag universal expression vectors named pCHAN and pCHAC were constructed in this study. At the same time, some restriction enzyme cutting sites were added in the vector sequences. Then the vectors could be used as universal fusion expression vectors, which had 7 common restriction enzyme cutting sites (PstⅠ, SpeⅠ, XbaⅠ, BamHⅠ, KpnⅠ, SmaⅠ, ApaⅠ or SalⅠ). To test and verify the reliability of these 2 vectors, a purple acid phosphatase gene named GmPAP14 (GenBank No.: JN967626) was linked with the above mentioned vectors. Then the recombinant vectors pCHAN-GmPAP14 and pCHAC-GmPAP14 were transformed into Arabidopsis. By PCR and DNA sequencing identification methods, the T2 transgenic Arabidopsis lines were obtained. And the Western blotting results showed that the hybridization protein bands were detected in transgenic Arabidopsis plants, while it was not detected in the wild-type control, indicating these 2 HA tag fusion vectors could integrate the objective protein stably in plant. So they (pCHAN and pCHAC) could be used as universal fusion expression vectors in plant protein purification and the other related studies in the future.