r-PA真核表达载体的构建与鉴定

用SV40和CaMV 35S两种启动子分别启动瑞替普酶(reteplase，r-PA)和标记基因bar基因，构建能在海带中表达外源基因的重组表达载体。通过PCR方法扩增CaMV35S启动子、bar基因和r-PA基因，将扩增的3个片段分别克隆到pGEM-T Easy Vector上。将CaMV片段亚克隆到pCAT3-control载体上得到重组质粒pCAT／CaMV；再将bar基因切下插入到CAT／CaMV上得到重组质粒pCAT／C-B，最后将rPA基因片段切下后插入到pCAT／C-B上获得重组质粒pSVrPA／C-B。PCR、酶切及测序结果均表明已成功扩增出CaMV 35S、r-PA和bar基因片段，并已顺次正确插入到真核表达载体PCAT3-control上，最终成功构建了能在海带中表达r-PA的真核表达载体，用基因枪法转入海带中表达，FAPA法测定得到有体外纤溶活性的阳性海带，为后续研究工作打下坚实的基础。

Construction and Identification of r-PA Gene Eukaryotic Expressing Vector

Recombinant expression vector was constructed in which the r-PA(reteplase, r-PA) and bar genes were inserted under the control of promoters of SV40 and CaMV 35S respectively. The PCR technique was used to amplify the fragments of promoter CaMV 35S, bar and r-PA genes, and then the amplified fragments were cloned into pGEM-T Easy Vector, respectively. Recombinant plasmid pCAT/CaMV was obtained by subcloning CaMV fragment into pCAT3-control vector and thereby bar gene fragment was inserted into pCAT/CaMV and recombinant plasmid pCAT/C-B was produced. The r-PA gene was cut down from pGEM/rPA and was finally transferred into pCAT/C-B resulting in the eukaryotic expressing vector pSVrPA/C-B. PCR, enzyme digestion and sequencing results demonstrated that the CaMV 35S, r-PA and bar gene fragments were all successfully amplified and were inserted into PCAT3-control vector. Using biolistic particle delivery system the plasmid was delivered into Laminaria gametophytic to express. Activity of the products wass detected by fibrin agarose plate assay. The construction mentioned above paved the way for the expression of r-PA in transgenic plants such as Laminaria Japonica.