启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较  

Efficiency Comparison of Promoters Ptac & PsbA Driving hG-CSF Expression in Anabaena sp. PCC 7120

作　　者：宁文艳 吴先敏[1] 王春梅[1] 陈伟东[1] 施定基[2] 

机构地区：[1]北京中医药大学中药学院生物制药系,北京100102 [2]中国科学院植物研究所,北京100093

出　　处：《微生物学杂志》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)

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.

关 键 词：鱼腥藻7120 人粒细胞集落刺激因子 PTAC PSBA 表达 

分 类 号：Q943.2[生物学—植物学]