瑞替普酶(reteplase)在海带中表达的鉴定分析

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1100bp处有特异性条带，Western blotting在39ku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-PAN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。

Identification of Reteplase Expressed in Laminaria Japonica

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 this study identified 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.