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
[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Met...[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.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 National 863 Project of China (2002AA227011)Natural Science Foundation of Hubei Province (2003ABAI18)Natural Science Foundation of Shandong Province (ZR2010HQ054)~~
文摘[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.