大鼠间质胶原酶哺乳动物表达质粒的构建和体外转基因表达

Construction of mammalian expression plasmid of Flag-tagged rat collagenase and its in vitro transfection study

目的构建大鼠间质胶原酶哺乳动物细胞表达质粒并进行体外转基因研究。方法采用ＰＣＲ及基因重组技术构建融台有Ｆｌａｇ多肽的大鼠间质胶原酶哺乳动物表达质粒，用脂质体介导该表达质粒体外转染培养的ＮＩＨ３Ｔ３细胞，通过ＲＴ－ＰＣＲ方法在转染细胞中检测间质胶原酶融合蛋白的ｍＲＮＡ表达，采用ｗｅｓｔｅｒｎｂｌｏｔ方法在转染细胞的培养上清液中检测间质胶原酶融台蛋白的分泌，采用明胶酶谱法检测表达的间质胶原酶的明胶降解活性。结果构建的融合Ｆｌａｇ的大鼠间质胶原酶哺乳动物表达质粒经脂质体介导体外转染ＮＩＨ３Ｔ３细胞后能够转录、表达并分泌间质胶原酶融合蛋白，并且，这一表达产物具有降解明胶的酶活性。结论我们成功构建了融合Ｆｌａｇ的大鼠间质胶原酶哺乳动物细胞表达质粒，体外转染研究证实该质粒能够在ＮＩＨ３Ｔ３细胞中表达活性问质胶原酶，为进一步运用该质粒进行体内转基因等研究奠定了基础。

Objective A mammalian expression plasmid of rat collag6nase was constructed andits expression in NIH3T3 cells in vitro was studied. Methods PCR and g6ne recombinant techniqueswere used to construct a mammalian expression plasmid of Flag-fusion rat collagenase. This plasmidwas then transferred into cultured NIH3T3 cells mediated by lipofectamine. The mRNA and proteinexpression of the Flag epitome tagged rat collagrnase were detected by RT-PCR and western blottechniques. The enzyme activity of expressed collagrnase was detected by grlatin zymography. RAfter transfection the mRNA expression of Flag-fusion rat collagrnase could be detected in NIH3T3cells and the secretion of this collagrnase could be detected in the culture medium, and this recombinant collag6nase kept the gelatin degradation activity. Conclusion This constructed plasmid canexpress active rat collag6nase in vitro and can be used for further study.