重组纤溶酶原Kringle 5区基因杆状病毒载体的构建及表达研究

目的克隆人源性纤溶酶原Kringle5(K5)基因,构建K5分泌型杆状病毒载体,并检测其在肌细胞中的表达。方法引物中加入前胰岛素原信号肽基因序列,PCR产物经Age Ⅰ和Acc Ⅰ双酶切后装入pFB载体中构建为pFBK5,将其转染肌细胞C2C12,经Western印迹检测重组蛋白的表达并用MTT试验进行初步活性鉴定。结果成功扩增出了K5基因序列,测序正确,转染了pFBK5的C2C12细胞及上清中均检测到了特异性的14 ku蛋白条带且其对ECV304细胞产生剂量依赖性抑制作用,而转染空载体对照组无相应蛋白表达。结论人源性纤溶酶原K5分泌型杆状病毒载体构建成功,表达的重组蛋白具有生物活性,为进一步的病毒制备奠定了基础。

Construction of Recombinant Human Plasminogen Kringle5 Baculovirus Vector and Secreted Expression in Myoblast

Objective To clone the human plasminogen kringle5 gene and construct pFBK5, then detect the expression of K5 in C2C12. Methods By using PCR technique to amplify the human plasminogen kringle5 gene, the gene of preprotrypsin leader was added to the primer for the secreted expression of K5 from C2C12. After sequencing, the product was inserted into the baculovir-us vector (pFB). Then pFBK5 was transfected into C2C12, and 24-48 hours later, the proteins were obtained from C2C12 and the supernatant. Afterwards, K5 was tested by using Western blot and the activity checked by MTT. Results The acquired k5 gene was 249 bp which encodes 83 a-mino acids, the protein of 14 ku. By using Western blot we detected the special 14 kU protein from the supernatant and C2C12 transfected with pFBK5, which inhibited the proliferation of ECV304. Whereas K5 was not found in those transfected with pFBGFPR. Conclusion pFBK5 was constructed successfully and the recombinant protein has activity.