pcDNA3.1+-MBL 真核表达载体构建及其在不同哺乳动物细胞株中的表达

目的　比较人甘露聚糖结合凝集素 (MBL)基因在不同哺乳细胞株中的表达效率。方法　应用PCR从含有中国人野生型MBLcDNA的质粒pGEM MBL中扩增目的基因片段 ,将其克隆至pcDNA3.1 表达载体 ,经酶切及测序鉴定后 ,以电穿孔法转染CHO、HEPG2和HEK2 93细胞 ,以RT PCR、ELISA分析其在 3株细胞中的表达情况。结果　从pGEM MBL中扩增得到约 75 0bp的MBLDNA片段 ,构建成重组表达载体pcDNA3.1 MBL ,经酶切出现 75 0bp片段 ,测序鉴定与预期的完全一致。将其转染进细胞后 ,经G4 18选择转染子并克隆化培养 ,RT PCR和ELISA分析显示 ,在CHO和HEK2 93细胞中及培养上清液中分别有较高的MBLmRNA和蛋白表达 ,而HEPG2细胞表达较低。结论　人MBL在CHO和HEK2 93细胞中的表达效率较高 ,为今后在哺乳动物细胞中高效表达人MBL积累了经验

Construction of eukaryotic expression vector pcDNA3.1+ -MBL and its expression in different mammalian cell lines

Objective To compare the expression efficiencies of human mannan-binding lectin (MBL) gene in different mammalian cells. Methods The target sequence in pGEM-MBL plasmid that contained human MBL cDNA was amplified by PCR, inserted into eukaryotic expression vector pcDNA3.1 , and then identified by restriction mapping and sequencing. The recombinant expression vector was transformed into Chinese-hamster ovary (CHO) cells, human hepatoma cell line (HEPG2), and human embryonic-kidney-cell line 293 (HEK293) by electroporation. The G418-resistant clones were selected as well as the MBL mRNA and the protein expression were analyzed by RT-PCR and ELISA, respectively. Results The cDNA fragment of about 750 bp was amplified from pGEM-MBL plasmid and the recombinant expression vectors PcDNA3.1 -MBL were constructed and transformed into CHO, HEPG2, and HEK293 cells successfully. Analyses of expression efficiencies of the selected G418-resistant clones showed that the expressions of MBL mRNA and proteins were higher in CHO and HEK293 cells than those in HEPG2 cells. Conclusion CHO and HEK293 cells can express human MBL in higher-efficiency, which provides the basis for further research of recombinant MBL expression in mammalian cells.