新型rPA(K)原核表达载体的构建及初步表达

The Construction and Expression of New-type rPA(K) Prokaryotic Vector

利用PCR技术,获得了rPA(K)cDNA片段,将其克隆至pET-30a(+),成功构建了新型的rPA(K)原核载体pLrA(K),并实现了其在大肠杆菌中的初步表达,经IPTG(终浓度为1 mmol/L)分别于37℃诱导1,2,3 h后,以Bio-Rad凝胶成像仪分析目的蛋白,相对含量分别是19%,15.8%和24.3%,平均密度分别是0.13,0.14和0.16,IPTG诱导3 h后蛋白表达量最高.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210μg/L.

By using PeR, the rPA（K） cDNA was got, then it was cloned into pET-30a （ ＋ ） to get me newtype rPA（K） prokaryotic vector and it was expressed successfully. The initial content of target protein went to 24.3 % and the average density was 0.16. The molecular weight was about 43 ku. The ELISA indicated the expressive product had specific positive response to anti-t-PA antibody and its average content was 210μg/L.