摘要
在原核表达载体pErA(K)的基础上,构建新型原核分泌表达载体pBrA(K),并实现其在原核表达系统中的表达。用限制性内切酶NcoI,XhoⅠ同时双酶切获得目的基因rPA(K)后,以T4连接酶将其连接到pET-20b(+)上,构建新型原核分泌表达载体pBrA(K)并实现了在大肠杆菌中表达,经纤维蛋白平板检测存在于重组菌株培养液上清中的分泌蛋白有溶纤活性。ELISA结果显示表达产物与抗t-PA抗体呈现特异性阳性反应,进一步参考标准曲线得出样品中rPA(K)的平均含量为51 ng/mL。实验结果证明新型原核分泌表达载体pBrA(K)构建成功,并实现了其在原核表达系统中的表达,为新型rPA(K)的表达优化及后续的纯化研究工作奠定了基础。
On the base of pErA（K）,pBrA（K） would be constructed and the expression in the prokaryotic secretory system would be achieved.with Nco I and Xho I used by T4 ligase.Through mFAPA,the secretory protein in supernatant had the fibrinolysis activity.ELISA showed that the expressive product had specific positive response with anti-t-PA antibody and its average content was 51 ng/mL.The results showed that pBrA（K） had been constructed successfully,and the expression in the prokaryotic secretory system was achieved.
出处
《生物学杂志》
CAS
CSCD
2010年第5期7-10,30,共5页
Journal of Biology
基金
宁夏自然科学基金资助(项目号:NZ0505)
