人肠三叶因子重组真核表达载体的构建及在CHO/dhfr-细胞中的稳定表达

目的：构建人肠三叶因子（hITF）重组真核表达载体，并检测其在CHO/dhfr-细胞中的表达。方法：通过重叠延伸PCR法获得hITF cDNA片段，将目的基因插入真核表达载体pIRES/dhfr，得到重组真核表达载体pIRES/hITF/dhfr；经脂质体介导转染CHO/dhfr-细胞，通过G418筛选，建立稳定转染的CHO/dhfr-细胞系；用RT-PCR及ELISA法检测hITF的表达。结果：成功构建了重组真核表达载体pIRES/hITF/dhfr，并建立了稳定转染的CHO细胞系，成功地表达了目的基因。结论：成功构建了重组真核表达载体pIRES/hITF/dhfr，并在CHO/dhfr-细胞中分泌表达hITF蛋白，为进一步进行重组hITF的临床前研究奠定良好的实验基础。

Construction of recombinant eukaryotic expression vector of human intestinal trefoil factor and its expression in CHO/dhfr- cells

Objective: To construct eukaryotic expressing vector of human intestinal trefoil factor and express hITF protein in dihydrofolate reductase-deficient Chineses hamster ovary cells (CHO/dhfr-). Methods: The full length cDNA fragment was amplified by overlap extension PCR, then inserted into eukaryotic expression vector pIRES/dhfr. The recombinant vector was transfected into CHO/dhfr- cells by LipofectamineTM2000. The stable transfected CHO/dhfr- cell line was then established by screening cultures with G418. RT-PCR and ELISA were used to detect the expression of the protein. Results: The eukaryotic expression vector was constructed successfully, stable transfected CHO cell line was established, and hITF protein was expressed successfully. Conclusion: The recombinant plasmid pIRES/hITF/dhfr is successfully constructed and it expresses the protein in the supernatant of the CHO/dhfr- cells, which is the basis for pre-clinical study.