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将α-心钠素(α-hANF)基因克隆到PUC_(18)载体上,经菌落原位杂交筛选鉴定出有α-hANF基因的克隆株。用BamⅡ和BamH Ⅰ消化并分离纯化得到含5′端部分α-hANF基因及PUC_(18)载体的F_1片段。用DNA合成仪合成3′端部分α-hANF基因的F_2接头,在基因3′端加入谷氨酸的密码子GAA,作为表达产物用内肽酶Glu-C专一裂解的位置,并去掉基因3′端的终止信号TGA,将原BamH Ⅰ接头改为EcoR Ⅰ接头。最后,将F_1和F_2片段与α-hANF基因酶促连接后进行转化,通过双酶切筛选出含有双拷贝α-hANF基因的转化子,并用双脱氧链终止法进行序列分析,证明核苷酸顺序正确。
Theα-atrial natriuretic factor (a-hANF) gene was cloned into PUC_ (18) vector. The cloned strain with α-hANF gene was identified by in situ hybridization screening. After digestion with Bam II and BamH Ⅰ, the F 1 fragment containing the 5 ’-end α-hANF gene and the PUC_ (18) vector was isolated and purified. The F 2 linker of the 3’-terminal α-hANF gene was synthesized by a DNA synthesizer. The codon GAA of glutamic acid was added to the 3’-end of the gene as a specific cleavage site for Glu-C of the expression product, and the gene was removed 3 ’end of the signal TGA, the original BamH Ⅰ adapter to EcoR Ⅰ adapter. Finally, the F_1 and F_2 fragments were ligated with the α-hANF gene and then transformed. The double-copy α-hANF transformants were screened by double enzyme digestion and sequenced by dideoxy chain termination method. It was proved that the nucleotides The order is correct.