摘要
为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。
In order to compare the efficiency of the exogenous promoter Ptac and the endogenous promoter PsbA to drive exogenous gene transcription in Anabaena sp. PCC 7120, two shuttle expression vectors pRL-PsbA-GCSF and pRL-Tac-GCSF were constructed. The vectors were transferred into the cyanobacteria by tri-parental conjugative transfer methods. The transgenic cyanobacteria cells were screened by antibiotic and determined through plasmid extraction and PCR assay and obtained the cyanobacteria of transgenic hG-CSF that expressed respectively drived by the two promoters, and the goal gene existed in the form of plasmid. The transcription levels of the hG-CSF in the two transgenic cyanobacteria were compared by semi-quantitative RT-PCR method and it was found that there was no difference between the two transgenic cyanobacteria drived with promoter PsbA and Ptac. The expression of the hG-CSF protein in transgenic cyanobacteria was compared by ELISA. It was found that the expression of hG-CSF promoted by PsbA was 1.17 times of that promoted by Ptac under induced conditions.
出处
《微生物学杂志》
CAS
CSCD
2014年第3期36-41,共6页
Journal of Microbiology
基金
北京中医药大学自主选题项目(2013-JYBZZ-JS-139)
作者简介
宁文艳 女，硕士研究生。研究方向为基因工程制药。E-mail：xiaonin@984@126．com
通讯作者。女，博士，教授。研究方向为基因工程制药。Tel：010—84738646，E-mail：wchunmei@126．com
