重组人β-防御素-2基因的植物表达载体的建立

Construction of Plant Expressive Vector of Human β-defensin-2 Gene

目的 运用植物转基因技术 ,探索构建表达人类 β-防御素 - 2 ( h BD- 2 )的植物生物反应器的可能性及技术路线。方法 将 C端带 myc和 6x His双标记的重组 h BD- 2基因插入植物真核表达载体 p CAMBIA13 0 4中 ,构建 C端带 m yc和 6x His双标记的重组 h BD- 2植物真核表达载体 rp CAMBIA13 0 4/ h BD- 2 / His。利用此载体转化根癌农杆菌 L BA44 0 4,经卡那霉素进行抗性基因筛选和 PCR检测 ,确定根癌农杆菌的阳性克隆。重组 h BD- 2通过转化阳性的根癌农杆菌被导入小麦幼胚愈伤组织细胞 ,经潮霉素进行抗性基因筛选 ,获得抗性愈伤组织。结果 酶切分析、PCR验证和 DNA测序等证据表明 :C端带 m yc和 6x His双标记的重组 h BD- 2已被正确地插入 p CAMBI-A13 0 4载体中 ,并位于 Ca MV 3 5 S与 Nos终止子之间 ,提示重组 h BD- 2 / His基因的植物表达载体 rp CAMBI-A13 0 4/ h BD- 2 / His已被成功构建。卡那霉素抗性基因筛选和 PCR检测显示 :rp CAMBIA13 0 4/ h BD- 2 / His已成功转化根癌农杆菌 L BA44 0 4,阳性克隆菌株已获得。潮霉素抗性筛选结果提示 :h BD- 2基因被导入小麦幼胚愈伤组织细胞。结论 本研究为进一步培育 h BD-

Objective Several evidences suggested that transgenic plants would be a facile and economic bioreactor for large-scale production of industrial and pharmaceutical recombinant proteins. This study is made in an attempt to establish plant bioreactor for expression of recombinant hBD-2. Methods Recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was inserted into a plant expressive vector-pCAMBIA1304, which closely located the down-stream of CaMV35S promoter. Agrobacterium tumefaciens LBA4404 was transformed with the recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His. The positive clones of LBA4404 transformed by rpCAMBIA1304/hBD-2/His were selected on a culture plate containing kanamycin. The callus tissues were transfected by positive clones of LBA4404, and positive callus were examined by using the resistant selection of hygromycin gene. Results The evidences of enzyme digestion, PCR and sequence analysis confirmed that recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was correctly inserted into pCAMBIA1304 and was located between CaMV35S promoter and Nos terminal cordon to construct recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His, thus indicating that rpCAMBIA1304/hBD-2/His has successfully transformed Agrobacterium tumefaciens LBA4404 and positive clones have been isolated. The results from resistant selection of hygromycin gene showed that rpCAMBIA1304/hBD-2/His has been transferred into the callus of wheat, and the differentiation of callus tissue under selective pressure of hygromycin is carried out continually. Conclusion The above data suggest that the technique of transgenic plant is workable for the production of recombinant hBD-2.