摘要
用PCR方法从海洋单细胞蓝藻聚球藻 70 0 2 (Synechococcussp .PCC 70 0 2 )基因组DNA中扩增得到藻蓝蛋白β亚基基因 (cpcβ)的上游序列 (Pcpcβ) ,及编码谷氨酰胺合成酶的 glnA基因片段 .以Pcpcβ作为启动子、以glnA基因片段作为整合平台 ,构建含有小鼠金属硫蛋白 Ⅰ (mMT Ⅰ )cDNA的同源整合表达载体 pKGC MT .通过自然转化法将整合表达载体导入聚球藻 70 0 2中 ,经氨苄青霉素筛选 ,得到遗传性状稳定的转基因藻 .PCR检测证明mMT Ⅰ基因已整合到蓝藻基因组DNA上 ;蛋白质印迹表明mMT Ⅰ已在蓝藻中表达 ;ELISA结果显示mMT Ⅰ在蓝藻中的表达量约为 80 0 μg/ g .
The 300 bp upstream fragment of cpcβ gene which encodes the β-subunit of phycocyanin and the 1.4 kb fragment of glnA gene encoding glutamine synthetase were obtained by polymerase chain reaction (PCR) from genomic DNA of marine cyanobacterium Synechococcus sp. PCC 7002. Then, integrative expression vector pKGC-MT, which contained promoter Pcpcβ, mMT-Ⅰ gene and integrative platform glnA, was constructed and introduced into Synechococcus sp. PCC 7002 via natural transformation. Selected by ampicillin, the stable transgenic cyanobacterium was obtained. PCR analysis indicated the integration of mMT-Ⅰ gene in genomic DNA of Synechoccus sp. PCC 7002 and Western blotting demonstrated the expression of mMT-Ⅰ in the cyanobacterium. According to the result of ELISA, the amount of the expressed mMT-Ⅰ was about 800 μg/g fresh cells.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2002年第1期149-153,共5页
Progress In Biochemistry and Biophysics
基金
国家"九五"攻关项目 (96 C0 2 0 4 0 5)~~
