瑞替普酶(reteplase)在海带中表达的鉴定分析

Identification of Reteplase Expressed in Laminaria Japonica

为验证本实验室构建的瑞替普酶(reteplase,r-PA)基因是否成功转入海带,本文通过FAPA、PCR、Southern blotting和Western blotting等方法分别从体外纤溶活性、DNA的整合以及目的蛋白的表达等三个方面进行鉴定,并运用固定化金属离子亲和层析对表达的蛋白进行了初步分离纯化。FAPA结果表明转基因海带蛋白具有溶栓活性,PCR和Southern Blotting显示在1 100 bp处有特异性条带,Western blotting在39 ku处有蛋白条带,证明r-PA基因已成功转入海带而且获得稳定表达,海带中表达的r-PA不需复性就具有溶栓活性,同时说明在r-PA N端设计的(His)6有助于我们利用亲和层析分离纯化目的蛋白。

In order to identify if the gene of reteplase（r-PA） has been successfully transferred into the La minaria Japonica , the r-PA was studied on the level of activity, the integration of DNA and expression of protein with the methods of FAPA, PCR, Southern blotting and Western blotting. At the same time, the expression r-PA protein was initially purified by the Ni-chelating chromatography. The results showed that the supernatant of the grinded La minaria Japonica has the activity of thrombolysis. PCR and Southern blotting displayed that there was a special bind at the position of 1100bp. SDS-PAGE and Western blotting pictured that there was a visible band at the position of 39KD. All these identified that the gene of reteplase had been successfully transformed into La minaria Japonica and had expressed stably, and the r-PA protein had the activity of Thrombolysis without the need of renaturation. It is also demonstrated in this paper that the （His）6 at the end of N-ter minal had an advantage to purify the r-PA protein easily.