摘要
目的构建以增强型绿色荧光蛋白(EGFP)为筛选标记的新型pUC18筛选载体。方法将EGFP基因克隆至pUC18载体的PstI/HindⅢ位点,构建筛选载体pUC18-EGFP,并验证pUC18-EGFP能否利用EG-FP的荧光特性筛选出含基因重组体的阳性克隆。结果经酶切和基因测序鉴定pUC18-EGFP载体构建正确;在外源基因插入该载体后,对LB氨苄平板上的绿色菌落进行酶切和基因测序鉴定,结果证明LB平板上的绿色菌落即为含有重组质粒的克隆。结论成功构建pUC18-EGFP筛选载体,利用EGFP的绿色荧光可筛选出阳性克隆。
Objective To construct and identify the s FP as an indicator. Methods The EGFP gene was creening prepared vector pUC18-EGFP, using EG- by PCR and cloned into pUC18 resulting the vector pUC18-EGFP. Then DNA fragment was inserted into the MCS of pUC18-EGFP to test its practicability based on green fluorescence. Results The pUC18-EG- FP was confirmed correctly by restriction enzyme analyses. The pUC18-EGFP was used to select recombinants. The green strains on the plate were confirmed by restriction enzyme and DNA analyses, which were E. coli harboring recombinants. Conclusion The screening vector PUC18-EGFP was constructed successfully. Thus, we can select the positive clones on plates based on the green fluorescence of EGFP.
出处
《中国海洋药物》
CAS
CSCD
2009年第2期28-31,共4页
Chinese Journal of Marine Drugs
基金
山东省科技攻关计划项目(2008GG10002022
2008GG20071000)
国家自然科学基金(30772497)
山东省卫生高层次人才1020工程专项经费
校青年教师科研基金(KQ07010)
关键词
增强型绿色荧光蛋白
大肠杆菌
阳性克隆
筛选载体
enhanced green fluorescent protein （EGFP）
E. coli
positive clone
screening vector
