通过对硅胶基质进行化学改性键合伴刀豆球蛋白(C on A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RN ase B...通过对硅胶基质进行化学改性键合伴刀豆球蛋白(C on A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RN ase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RN ase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALD I-TO F M S)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RN ase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。展开更多
AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell...AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2 000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.展开更多
文摘通过对硅胶基质进行化学改性键合伴刀豆球蛋白(C on A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RN ase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RN ase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALD I-TO F M S)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RN ase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。
文摘【目的】探讨Dicer和Drosha在子宫内膜癌的发生发展中的作用及意义。【方法】利用荧光定量PCR和Western‐blot检测正常子宫内膜组织和子宫内膜癌组织中Dicer和Drosha的表达；体外培养正常子宫内膜细胞，采用siRNA干扰Dicer和Drosha ，M T T检测子宫内膜细胞的增殖；流式细胞术检测细胞凋亡；Western‐blot检测凋亡抑制蛋白Bcl‐2和促凋亡蛋白Bad的表达。【结果】子宫内膜癌组织中Dicer和Drosha的表达低于正常子宫内膜组织；siRNA干扰Dicer和Drosha后子宫内膜细胞的增殖加快、凋亡减少；Bcl‐2表达增加而Bad的表达减少。【结论】Dicer和Drosha可以影响细胞的增殖和凋亡，其相关机制可能与调控Bcl‐2和Bad的表达有关，Dicer和Drosha在子宫内膜癌的发生发展中起重要作用。
文摘AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2 000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.
