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Endostar体外稳定性研究

In vitro stability of Endostar

  • 摘要: 目的 : 比较重组人内皮抑素恩度 TM (Endostar TM )和毕赤酵母表达的内皮抑素(endostatin)在不同温度及不同pH条件下的稳定性,通过检测对内皮细胞的增殖抑制比较Endostar在4 ℃和37 ℃下的生物活性。 方法 : 将Endostar和endostatin于4 ℃保存或37 ℃放置96 h后进行不同条件的聚丙烯酰胺凝胶电泳,比较Endostar和endostatin之间以及它们在不同温度放置后的电泳行为,采用兔源多克隆抗体进行Western blot验证。同时检测在两种温度下的Endostar对于人脐静脉内皮细胞HUVEC细胞增殖的抑制作用。 结果 : 在还原性电泳中Endostar和endostatin在20 kD左右处均只出现一条带。在3种酸性非还原电泳条件下,endostatin出现明显的“smear”现象,无法观察到主要条带,Endostar在酸性不连续非还原电泳条件下只出现一条带,而在两种连续非还原电泳中虽然也出现“smear”现象,但是主要条带仍然存在,Endostar和endostatin在37 ℃放置96 h后与它们保存在4 ℃的样品相比,在各种电泳条件下有相似的电泳行为,Western blot验证了相应电泳的结果。在两种温度下的Endostar对于HUVEC的增殖抑制作用基本相同。 结论 : Endostar和endostatin具有相似的热稳定性,但在酸性条件下Endostar较endostatin更为稳定。

     

    Abstract: Aim :To compare the stability of EndostarTM and endostatin under different temperatures and pH using polyacrylamide gel electrophoresis(PAGE) and Western blot and to compare the activity of Endostar kept at 4 ℃ and 37 ℃ by inhibition of endothelial cell proliferation. Methods :Endostar and endostatin expressed by Phicha pastoris were kept at 4 ℃,and 37 ℃ for 96 hours,respectively.The electrophoresis of the samples was detected by reduced and non-reduced PAGE.The results were further confirmed by Western blot with rabbit anti-Endostar polyclonal antibody.The inhibitory effect of Endostar stored at different temperatures on HUVEC was also examined by cell-based assay. Results :Single band at 20 kD was detected in all lanes of SDS-PAGE gel loaded with endostatin and Endostar samples under reducing condition.In acidic native PAGE with three different pH values,endostatin showed a “smear” characteristic,whereas Endostar showed a unique band in acidic non-continuous native PAGE.Although the “smear” phenomenon was also observed under two conditions of constant native electrophoresis,the major band of Endostar could be detected.Similar electrophoretic behavior was found for endostatin and Endostar stored at both 37 ℃ and 4 ℃.Western blot showed similar results to those by PAGE.Furthermore,Endostar stored at these two temperatures also had identical inhibitory effect on proliferation of HUVEC. Conclusion :Endostar and endostatin exhibit similar thermostability at regular conditions,but Endostar was more stable than endostatin expressed in P.pastoris under acidic condition.

     

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