鲎抗脂多糖因子的基因克隆及其在COS-7细胞中的表达

Cloning and expression in COS-7 cells of limulus Anti-Lipopolysaccharide factor Gene from Chinese Horseshoe Crab

目的构建中国鲎的鲎抗脂多糖因子(LALF)基因序列的真核表达载体并尝试其在真核细胞COS-7中的表达。方法从鲎血细胞中提取mRNA,经RT-PCR扩增出LALF的基因片段,将其克隆入真核表达载体pcDNA3.1/myc-His(-)中进行酶切鉴定和序列测定,重组载体通过脂质体转染COS-7细胞进行表达,SDS-PAGE分析表达产物。结果酶切鉴定和序列分析证实重组质粒含有LALF的基因片段。经RT-PCR证实转染的COS-7细胞含有LALF的基因序列,经SDS-PAGE分析表明转染重组质粒的COS-7细胞表达融合蛋白。结论LALF真核表达载体构建成功并实现其在真核细胞COS-7中的表达,为进一步探讨rLALF的生物活性奠定了基础。

To construct the eukaryotic expression vector containing native LALF(Limulus anti lipopolysaccharide factor) gene from Chinese horseshoe crab and attempt to express the target protein in COS-7 cellls.The mRNA was extracted from amebocytes of Chinese horseshoe crab and the LALF gene sequence was synthesized and amplfied by RT-PCR. The PCR product was cloned into pcDNA3.1/myc-His(-) plasmid and the DNA sequence was examined by restriction enzyme digestion and DNA sequence analysis. The recombinant plasmid was transfected into COS-7 cells by lipofectamine for expression and the positive clones were identified by SDS-PAGE.The LALF gene sequence was confirmed to be present in recombinant plasmid and the transfected COS-7 cells.And the expression of recombinant protein was shown by SDS-PAGE analysis. The construction of eukaryotic expression vector of LALF gene from Chinese horseshoe crab and its expression in COS-7 cells were both successful, which is helpful to the further study on the biological activity of rLALF.