单顺反子表达人GM-CSF和B7.1融合基因真核表达载体的构建和鉴定

Construction and Identification of Eukaryotic Vector Expression Containing Human GM-CSF and B7.1 Fusion Gene from A Single Cistron

目的 构建单顺反子表达人 GM- CSF和 B7.1融合基因真核表达载体。 方法 应用巨引物 PCR技术对 h GM- CSF基因 c DNA进行定点突变 ;应用 PCR重叠延伸法 ,将 h GM- CSF和 h B7.1基因的 c DNA编码序列通过一 linker序列拼接 ,构建 h GM- CSF与 h B7.1融合基因 h GM- B7.1,将其插入 pc DNA3真核表达载体 ,测定核苷酸序列。 结果 序列分析表明 :(1) h GM- CSF突变体基因 c DNA编码序列的 178位核苷酸由 C变为 A,其余核苷酸序列均未发生变化 ;(2 ) h GM- CSF、linker、h B7.1的连接顺序、方向及序列完全正确。 结论 构建 pc DNA3- h GM- B7.

Objective To construct eukaryotic vector expression with human GM\|CSF and B7.1 fusion gene containing a single cistron. Methods Human GM\|CSF cDNA was made point mutation by megaprimer PCR. Human GM\|CSF and B7.1 cDNA were spliced through a linker sequence by over\|lap extention PCR. The fusion gene was inserted into pcDNA3 eukaryotic expressing plasmid and its sequence was analyzed. Results Sequence analysis showed that the coding sequence of hGM\|CSF cDNA was mutated from C to A at 178 nucleotide and the splicing order, the direction and the sequence in hGM\|B7.1 fusion gene were all correct. Conclusion hGM\|B7.1 fusion gene was successfully constructed.