国家“九五”攻关项目(96-C02-04-05).
This work was supported by a grant from the National “The Ninth Five” Project (96-C02-05).
用PCR方法从海洋单细胞蓝藻聚球藻7002(Synechococcus sp. PCC 7002)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7002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800 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.
周杰,罗娜,宁叶,施定基,俞梅敏,茹炳根.通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究[J].生物化学与生物物理进展,2002,29(1):149-153
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