【摘要】 利用RT-PCR技术从NIH 3T3细胞中克隆出环氧化酶ⅡcDNA,全长1815bp,测序分析表明与文献报道已知相符,构建真核表达载体。用阳离子脂质体介导转染的方法转染至宿主细胞HEK293细胞中,G418筛选出稳定转染的细胞克隆。加入底物花生四烯酸,通过测定PGE2含量的变化判断酶活,结果表明重组质粒能够表达环氧化酶。更多还原

【Abstract】 To obtain the full-length cDNA clone for cyclooxygenase II, we applied RT-PCR to amplify the cDNA from the NIH 3T3 cells. The resulting ISOObp fragment was cloned into the plasmid vector pcDNA 3.1 ( + ) and sequenced. The sequence was identical with the Gene Bank. The transfection of target DNA into HEK 293 cells was performed with the protocol using lipofectin reagent. We used the G418 to screen the stable cell lines, then assayed the content of PGE2 to identify the enzyme activity.更多还原