【摘要】 将人尿激酶原(pro-UK)cDNA分别插入家蚕核型多角体病毒转移载体pBK283和pBF4中,构建成两个重组质粒。所构建的这两个重组质粒与野生型家蚕核型多角体病毒DNA(BmNPV genomic DNA)共转染家蚕培养细胞,经病毒斑实验(Plaque assay)筛选出含pro-UK cDNA的稳定重组病毒株BmNPV-pk1和BmNPV-pk2。将此两株重组病毒分别感染家蚕培养细胞和家蚕幼虫4天后,用酶联免疫测定法(ELISA),纤维蛋白平板溶圈测活法和Western印迹法分析细胞培养上清及细胞和家蚕的体液及组织,证实均有pro-UK表达。家蚕培养细胞的表达量分别为0.0012mg/mL(上清液和10~6细胞)和0.0031mg/mL(上清液和10~6细胞),家蚕幼虫体液的表达量分别为0.0100mg/mL和0.0300mg/mL。从以上结果可看出,家蚕幼虫体液的表达量是培养细胞的10倍,以上表达产物在纤维蛋白平板上测活均有溶纤活性。更多还原

【Abstract】 Human pro-UK cDNA was inserted into Bombyx mori nuclear polyhedrosis virus (BmNPV) transfer vectors pBK283 and pBF4. The resulting plasmids and the BmNPV genomic DNA were co-transfected into Bm-N cells, and two stable recombinant viruses BmNPV-pk1 and BmNPV-pk2 were isolated by plaque assay on Bm-N cells. When Bm-N cells and silkworm larvae were infected with recombinant viruses BmNPV-pkl and BmNPV-pk2, the samples of cells fluid and silkworm larvae haemolymph were characterized with Western blot analysis, the blot displayed a major band at 50kDa. Enzymatic acticity in fibrin plate assay was 120IU per mL of cell supernatant. The expression level of pro-uk in the cells fluid 4 days post-infection was 3.1μg/mL, and in infected larvae haemolymph was 30.0μg/mL determined by ELISA.更多还原