目的克隆 HL A- E c DNA,并使其在 HL A 类阴性的靶细胞 L c L72 1.2 2 1细胞上获得稳定表达。方法用 RT-PCR方法从人外周血淋巴细胞扩增出 HL A - E c DNA ,并通过内核糖体进入位点 (IRES)将目的基因亚克隆于已经载有 HL A-A2的逆转录...目的克隆 HL A- E c DNA,并使其在 HL A 类阴性的靶细胞 L c L72 1.2 2 1细胞上获得稳定表达。方法用 RT-PCR方法从人外周血淋巴细胞扩增出 HL A - E c DNA ,并通过内核糖体进入位点 (IRES)将目的基因亚克隆于已经载有 HL A-A2的逆转录病毒表达载体 p GCEN上 ,构建成 HL A - A 2 / E多顺反子表达载体 (p G/ A2 E) ,采用感染的方法将重组质粒转入L c L 72 1.2 2 1细胞 ,最后经 G418筛选及有限稀释 ,利用抗 HL A- E特异的单克隆抗体 3D12进行 FACS检测 ,以观察 HL A - E分子在靶细胞表面的表达情况。结果 HL A- E分子在经 p G/ A2 E转染的靶细胞表面获得明显的表达 (88.79% ) ,且显著高于表达 HL A- E的对照细胞株 JAR(2 6 .2 1% ) ,而经 p G/ A2载体转染的靶细胞则未获得表达。结论成功构建了 p G/ A2 E多顺反子表达载体 ,并使 HL A- E分子在 HL A 类阴性的 L c L72 1.2 2展开更多
The present study was designed to investigate the effects of start codon of nosM on the biosynthesis of nosiheptide. Target genes were amplified by overlap PCR. After homologous recombination to construct engineered s...The present study was designed to investigate the effects of start codon of nosM on the biosynthesis of nosiheptide. Target genes were amplified by overlap PCR. After homologous recombination to construct engineered strains, nosiheptide production was analyzed by HPLC. Three mutants with different start codon ofnosMwere constructed, and nosihcptide production of each mutant was analyzed and compared. Replacement of the start eodon of nosM significantly decreased the production of nosiheptide. In conclusion, start codon usage could greatly affect the biosynthetic efficiency in the biosynthetic gene cluster of nosiheptide.展开更多
文摘目的克隆 HL A- E c DNA,并使其在 HL A 类阴性的靶细胞 L c L72 1.2 2 1细胞上获得稳定表达。方法用 RT-PCR方法从人外周血淋巴细胞扩增出 HL A - E c DNA ,并通过内核糖体进入位点 (IRES)将目的基因亚克隆于已经载有 HL A-A2的逆转录病毒表达载体 p GCEN上 ,构建成 HL A - A 2 / E多顺反子表达载体 (p G/ A2 E) ,采用感染的方法将重组质粒转入L c L 72 1.2 2 1细胞 ,最后经 G418筛选及有限稀释 ,利用抗 HL A- E特异的单克隆抗体 3D12进行 FACS检测 ,以观察 HL A - E分子在靶细胞表面的表达情况。结果 HL A- E分子在经 p G/ A2 E转染的靶细胞表面获得明显的表达 (88.79% ) ,且显著高于表达 HL A- E的对照细胞株 JAR(2 6 .2 1% ) ,而经 p G/ A2载体转染的靶细胞则未获得表达。结论成功构建了 p G/ A2 E多顺反子表达载体 ,并使 HL A- E分子在 HL A 类阴性的 L c L72 1.2 2
基金supported by the grants from the"111"Project from the Ministry of Education of China and State Administration of Foreign Export Affairs of China(No.111-2-07)the National Key Project on Science and Technology of China(No.2012ZX09103101-030&2012ZX09201101-012)+3 种基金National Science Foundation of China(No.81172967)the Doctoral Fund from the Ministry of Education of China(No.20110096110011)the Science&Technology Pillar Program of Jiangsu Province(No.SBE201371217)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘The present study was designed to investigate the effects of start codon of nosM on the biosynthesis of nosiheptide. Target genes were amplified by overlap PCR. After homologous recombination to construct engineered strains, nosiheptide production was analyzed by HPLC. Three mutants with different start codon ofnosMwere constructed, and nosihcptide production of each mutant was analyzed and compared. Replacement of the start eodon of nosM significantly decreased the production of nosiheptide. In conclusion, start codon usage could greatly affect the biosynthetic efficiency in the biosynthetic gene cluster of nosiheptide.
