根据GenBank中已登录的猪旋毛虫49 ku ES抗原基因序列设计了1对引物,用SDS裂解液配合蛋白酶K方法从猪旋毛虫不同发育时期(成囊前期幼虫、肌幼虫、成虫)虫体中提取总RNA,用RT-PCR方法扩增49 ku ES抗原基因,将目的基因定向克隆入pMD18-T载...根据GenBank中已登录的猪旋毛虫49 ku ES抗原基因序列设计了1对引物,用SDS裂解液配合蛋白酶K方法从猪旋毛虫不同发育时期(成囊前期幼虫、肌幼虫、成虫)虫体中提取总RNA,用RT-PCR方法扩增49 ku ES抗原基因,将目的基因定向克隆入pMD18-T载体,转化大肠埃希氏菌TG1。用PCR、限制性内切酶EcoRⅠ和BamHⅠ进行单、双酶切鉴定,阳性质粒的测序结果表明,成功克隆了猪旋毛虫不同发育时期虫体49 ku ES抗原基因。序列分析结果显示:49 kuES抗原基因大小为948 bp,基因的保守性很强,序列同源性比较高,不同发育时期之间的差异很小。展开更多
Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were stud...Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E. coli as template. The Sj26GST cDNA was cloned into the downstream of human M. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E. coli Mycobacteria shuttle plasmid pBCG 2000 to construct the expression shuttle plasmid pBCG Sj26. The recombinant BCG and M. smegmatis mc 2 155, which were electroplated with pBCG Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15 % and 10 % of total bacterial protein in BCG and M. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.展开更多
The 10 ku zein gene of maize was under control of patatin class I promoter of potato and transferred into potato genome by the leaf-disc method. The expression of 10 ku zein was determined in the tuber of transgenic p...The 10 ku zein gene of maize was under control of patatin class I promoter of potato and transferred into potato genome by the leaf-disc method. The expression of 10 ku zein was determined in the tuber of transgenic plants by RT-PCR. Furthermore, the sulphur-containing amino acids in transgenic tuber increase remarkably.展开更多
文摘根据GenBank中已登录的猪旋毛虫49 ku ES抗原基因序列设计了1对引物,用SDS裂解液配合蛋白酶K方法从猪旋毛虫不同发育时期(成囊前期幼虫、肌幼虫、成虫)虫体中提取总RNA,用RT-PCR方法扩增49 ku ES抗原基因,将目的基因定向克隆入pMD18-T载体,转化大肠埃希氏菌TG1。用PCR、限制性内切酶EcoRⅠ和BamHⅠ进行单、双酶切鉴定,阳性质粒的测序结果表明,成功克隆了猪旋毛虫不同发育时期虫体49 ku ES抗原基因。序列分析结果显示:49 kuES抗原基因大小为948 bp,基因的保守性很强,序列同源性比较高,不同发育时期之间的差异很小。
文摘目的:对Ro 60在胃癌多药耐药中的功能进行研究．方法:克隆Ro 60编码基因,构建Ro 60编码基因的正义真核表达载体,将其转导入SGC7901 细胞,应用半定量RT-PCR技术,对基因转染细胞进行鉴定,通过MTT法进行体外药物敏感性分析,借助流式细胞仪检测细胞内蓄积的阿霉素．结果:成功构建了Ro 60正义真核表达载体, 应用脂质体介导法将其转导入SGC7901,Ro 60真核表达载体转染SGC7901细胞后其表达量明显增加,体外药物敏感性试验提示其对长春新碱、5-氟尿嘧啶、丝裂霉素、顺铂、阿霉素的敏感性减低,SGC7901细胞的IC50值 (mg/L)与未转染细胞和转染空白载体细胞相比,显著增加(2．28±0．11 vs 0．45±0．04,0．65 ±0．05;2．89±0．14 vs 0．61±0．21,0．90±0．11; 1．92±0．03 vs 0．54±0．03,0．75±0．21;1．41± 0．06 vs 0．45±0．03,0．54±0．03;0．28±0．03 vs 0．14±0．01,0．14±0．01;均P<0．01),细胞内阿霉素蓄积显著减少(56．30±2．49 mg/L vs 92．83 ±3．63,87．38±2．94 mg/L,P<0．01)．结论:Ro 60真核表达载体转染SGC7901细胞后显示了一些多药耐药的特性,提示Ro 60在胃癌多药耐药中发挥了一定的作用．
文摘Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E. coli as template. The Sj26GST cDNA was cloned into the downstream of human M. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E. coli Mycobacteria shuttle plasmid pBCG 2000 to construct the expression shuttle plasmid pBCG Sj26. The recombinant BCG and M. smegmatis mc 2 155, which were electroplated with pBCG Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15 % and 10 % of total bacterial protein in BCG and M. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.
基金the National High-Tech Project (Grant No. 010-04-03-01).
文摘The 10 ku zein gene of maize was under control of patatin class I promoter of potato and transferred into potato genome by the leaf-disc method. The expression of 10 ku zein was determined in the tuber of transgenic plants by RT-PCR. Furthermore, the sulphur-containing amino acids in transgenic tuber increase remarkably.
