用同源重组法将人肝金属硫蛋白突变体ββ基因整合在集胞藻6803中表达

The Integration and Expression of ββ Mutant Gene of Human Liver Metal lothionein in Synechocystis sp. PCC 6803 by Homology Recombination

将集胞藻 (Synechocystissp .)PCC 6 80 3上psbB的一段序列 (从 # 6 93bp到 # 2 5 6 3bp)插入pBluescriptKS的多克隆位点 ,构建带有整合平台的载体pKSB。再将人工合成的 ββ基因插入中间载体pRL_439上启动子PpsbA的下游 ,并将pRL_ββ上PpsbA和 ββ基因插入pKSB构建成整合表达载体pKSB_ββ。利用自然转化将整合表达载体导入集胞藻 6 80 3,并通过单交换同源重组使 ββ基因整合到集胞藻 6 80 3基因组DNA上。逐步提高氨苄青霉素浓度 ,筛选得到遗传性状稳定的转基因集胞藻 6 80 3。PCR检测转基因集胞藻 6 80 3,结果证实 ββ基因已整合到集胞藻 6 80 3的染色体上 ;Westernblotting结果表明 ,ββ基因在转基因集胞藻 6 80 3细胞中得到表达 ,ELISA测定表明在 5 0 μmol/L的Zn2 +诱导下 ββ在新鲜集胞藻 6 80 3中的蛋白表达量为 0 .739mg/g ;重金属耐受性实验表明 ,得到能耐受Cu2 +的转基因集胞藻 6 80 3。经原子吸收光谱法测定 ,转基因集胞藻 6 80 3在含低浓度Cu2 +(10 μmol/L)的培养液中对Cu2 +的去除率可达到 82 %

The integration plasmid pKSB was constructed by inserting a special recombination target, psbB, into multiple cloning site (MCS) of pBluescript KS. The fragment psbB is part of psbB gene (from #693 bp to #2?563 bp) from Synechocystis sp.PCC 6803. Human liver metallothionein mutant gene ββ chemically synthesized was inserted into downstream of the promoter PpsbA on the intermediary vector pRL_439. The expression vector pKSB_ ββ was constructed by inserting PpsbA as well as ββ gene into MCS of pKSB and was transferred into Synechocystis sp. PCC 6803. The stable transgenic strain has been obtained by raising concentration of ampicillin gradually in the process of selection. The results of PCR analysis indicated that ββ gene has been integrated in the Synechocystis sp. PCC 6803 chromosome DNA by single_crossover events. Western blotting and ELISA analysis showed that the ββ gene was expressed in the transgenic strain with an amount of 0.739 mg/g fresh cells. Cu 2+ tolerance experiments proved that the transgenic strain had higher Cu 2+ _resistance. Atomic absorption demonstrated that 82% of Cu 2+ could be removed by the transgenic strain when cultured in BG11 medium containing 10 μmol/L Cu 2+ for 3 d.