增强型绿色荧光蛋白(enhance green fluorescent protein, eGFP)基因是遗传操作系统中常用的报告基因,以eGFP为报告基因评价不同启动子在乳酸乳球菌(Lactococcus lactis)NZ9000中的调控能力,寻找能够高效表达外源蛋白的强启动子。以实验室构建的携带eGFP基因的pIB184-eGFP质粒为载体,PCR扩增来源于pLpCas9-0537、pMG36e、pNZ44的启动子P11、P32、P44,将其分别克隆至pIB184-eGFP上,构建基于eGFP的报告系统。酶标仪定量测定eGFP的表达强度,比较不同启动子的转录活性。结果表明,在乳酸乳球菌中启动子P11转录的活力十分微弱,启动子P23、P32、P44的转录活性分别比P11高约23.9、7.8、4.1倍。该文利用基于eGFP的报告系统,筛选出适用于乳酸乳球菌中不同转录活性的启动子,为外源基因在乳酸乳球菌中的表达奠定了基础。
The convenience and high efficiency make the enhanced green fluorescent protein (eGFP) widely used as a reporter in genetic manipulation. In order to screen strong promoters with high transcriptional activity in Lactococcus lactis NZ9000, a report system based on eGFP was constructed. Four promoters, including P11, P23, P32 and P44 were compared. The fragments of promoters P11, P32 and P44 were amplified by PCR using pLpCas9-0537, pMG36e or pNZ44 as template and cloned into pIB184-eGFP, respectively. The transcriptional activity of different promoters was compared by quantitative assay of eGFP using microplate reader. Quantitative assay of the fluorescence intensity showed the transcription activity of P11 was weak, and that of P23, P32 and P44 was 23.9, 7.8 and 4.1 times higher, respectively. Taken together, promoters with different transcriptional activity were screened out using the eGFP-based reporting system, which could be used as a basic tool for heterogenous gene expression in L. lactis.
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