人重组pEGFPC1-uPA ATF质粒的构建及表达的研究

Construction and expression of recombinant eukaryotic expression vector pEGFPC1-uPA ATF

目的构建含人uPA ATF基因的真核表达质粒,并研究其对小鼠细胞株Pam212细胞的影响。方法设计引物,利用PCR从原核表达质粒中扩增人uPA ATF基因片断,并导入真核表达载体的绿色荧光蛋白pEGFPC1中,利用电泳和测序检测构建状况,荧光显微镜观察和免疫细胞化学检测转染情况,通过MTT、体外迁移实验检测对小鼠Pam212细胞株的影响。结果电泳和测序表明构建的质粒准确,uPA ATF cDNA阅读框完整,连接部位序列正确;质粒转染Pam212细胞株成功,并且能抑制细胞的增殖和迁移。结论成功构建了真核表达载体pEGFPC1-uPA ATF质粒,明确了人uPA ATF基因片断能降低小鼠细胞的增殖和迁移,为进一步在体实验研究打下了基础。

Objective To construct eukaryotic expression plasmid of pEGFPCI-uPA ATF gone, and investigate the effect of the plasmid on proliferation and migration of mouse Pam 212 cells. Methods Human uPA ATF cDNA was cloned from the prokaryotic plasmid by PCR technique, and then inserted into the eukaryotic expression plasmid pEGFPC1. The reconstructed plasmid was transfected into Pam 212 cells, and then identified by fluorescent microscopy and immunocytochemistry. Proliferation and the migration of the transfected cells were explored. Retmlts The reconstructed plasmid of pEGFPCI-uPA ATF was confirmed by the sequence analysis. The reconstructed plasmid could reduce the proliferation and migration ability of Pare 212 cells. Conclusion The pEGFPCI-uPA ATF plasmid is constructed successfully, uPA ATF can reduce the growth and invasion of Pam 212 cells, which lay a foundation for further studies of uPA ATF in vivo.