Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the prod...Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 μg/106 cells/24 h in the presence of 5×10-6 mol/ L MTX, and the levels can be further raised to 3. 5-4 μg/106 cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.展开更多
【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为...【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。展开更多
FLOWERING LOCUS T(FT)是调控植物开花途径中的关键基因,前期研究中,在比较晚开花wym-97和早开花cx-49两个品种的BcFT启动子时,发现cx-49的BcFT启动子存在一个1577 bp的插入片段。为探索该插入片段对不结球白菜开花的影响,本研究构建不...FLOWERING LOCUS T(FT)是调控植物开花途径中的关键基因,前期研究中,在比较晚开花wym-97和早开花cx-49两个品种的BcFT启动子时,发现cx-49的BcFT启动子存在一个1577 bp的插入片段。为探索该插入片段对不结球白菜开花的影响,本研究构建不结球白菜酵母杂交cDNA文库,继而利用酵母单杂交技术筛选与BcFT启动子互作的蛋白。结果显示,本研究所得cDNA文库的库容为1.8×10^(6)CFU,重组率为100%,插入片段长度分布范围约400~2000 bp,平均长度大于1000 bp,表明不结球白菜酵母杂交cDNA文库构建成功。自激活试验表明,800 ng·mL^(-1)的金担子素(AbA)可以抑制诱饵载体的自激活。通过酵母单杂交试验筛选到结合BcFT启动子插入片段的上游蛋白BcSAP18和BcDEAR2。综上,本研究成功构建了不结球白菜酵母杂交cDNA文库并获得了BcFT启动子插入片段的互作蛋白,为进一步研究BcFT在不结球白菜开花中的调控机制奠定了基础。展开更多
Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase （pro-UK） on the stability in culture supematant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302...Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase （pro-UK） on the stability in culture supematant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr-CHO cells, and serum-free culture supematant was harvested and incubated at 4℃ and 37℃, respectively. The pro-UK activity in culture supematant was measured by the optical density （OD） increase with time （ 12 hours） at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured. Rcsults After 48 hours of incubation at 4℃, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37℃ decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37℃. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4℃ and 37℃. Conclusion Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supematant.展开更多
Ischemic stroke is most commonly caused by vascular occlusion due to thrombosis or arterial embolism. Recently, thrombolysis has been used with increasing frequency for the treatment of acute ischemic stroke. Among th...Ischemic stroke is most commonly caused by vascular occlusion due to thrombosis or arterial embolism. Recently, thrombolysis has been used with increasing frequency for the treatment of acute ischemic stroke. Among the drugs used for thrombolysis, only recombinant tissue plasminogen activator is widely accepted internationally （Albers et al., 2008）. In China, urokinase has been widely used for thrombolysis after acute ischemic stroke. Pro-uro- kinase is the precursor of urokinase. Compared with urokinase, pro-uroki- nase has greater ability to dissolve thrombus and is safer to use.展开更多
基金Project supported by the National High-Technology Program "863".
文摘Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 μg/106 cells/24 h in the presence of 5×10-6 mol/ L MTX, and the levels can be further raised to 3. 5-4 μg/106 cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.
文摘【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。
文摘Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase （pro-UK） on the stability in culture supematant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr-CHO cells, and serum-free culture supematant was harvested and incubated at 4℃ and 37℃, respectively. The pro-UK activity in culture supematant was measured by the optical density （OD） increase with time （ 12 hours） at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured. Rcsults After 48 hours of incubation at 4℃, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37℃ decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37℃. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4℃ and 37℃. Conclusion Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supematant.
文摘Ischemic stroke is most commonly caused by vascular occlusion due to thrombosis or arterial embolism. Recently, thrombolysis has been used with increasing frequency for the treatment of acute ischemic stroke. Among the drugs used for thrombolysis, only recombinant tissue plasminogen activator is widely accepted internationally （Albers et al., 2008）. In China, urokinase has been widely used for thrombolysis after acute ischemic stroke. Pro-uro- kinase is the precursor of urokinase. Compared with urokinase, pro-uroki- nase has greater ability to dissolve thrombus and is safer to use.
