摘要
应用RT-PCR技术,从人黑色素瘤Bowes细胞株中扩增出人组织型纤溶酶原激活剂(tis-sue-type plasminogen activator,t-PA)cD-NA。序列分析证实,与国外的报道完全一致。将含完整阅读框架的人t-PA cDNA克隆至昆虫细胞表达转移载体pBacPAK8中,获得重组质粒pBac-tPA。应用脂质体共转染法,将pBac-tPA和线性化杆状病毒BacPAK6 DNA共转染Tn-5B-1昆虫细胞。经空斑筛选获得11株重组病毒。经PCR鉴定与生物活性测定,获得一株高效表达t-PA的重组病毒BactPA3。在含胎牛血清的培养基中,t-PA表达活性在感染(MOI≈10)后72h左右达到最高,为3.04×10~3IU/ml,即相当于1.8×10~4IU/10~6细胞;在无血清培养基中,t-PA最高表达水平相差不大,但时间为感染后132h左右。经SDS-PAGE纤维蛋白自显影分析,分子量为68kDa左右。与从人黑色素瘤细胞培养液中提纯的天然t-PA相比,其受纤维蛋白原激活的特性、和纤维蛋白的亲和力及在血浆中的失活速率基本相同。表达的t-PA在血液循环中的半衰期为7min。
With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAKS. The constructed pBac-tPA and the linear BacPAKG virus DNA were cotransfected with Tn-5B-l insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPAS, was selected by PCR identification and biological activity comparison. The t-PA activity expressed
in serum-containing media reach the highest level at 72h postinfection. The activity was 3. 04×103IU/ml, e. g. 1. 8×104IU/106cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132h postinfection ). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68kDa. Its stimulation by fibrinogen, affinity with fibrin and inactiva-tion in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.
出处
《实验生物学报》
CSCD
2000年第4期293-300,共8页
Acta Biologiae Experimentalis Sinica
