摘要
将α-心钠素（α-hANF）基因克隆到PUC<sub>18</sub>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<sub>18</sub>载体的F<sub>1</sub>片段。用DNA合成仪合成3′端部分α-hANF基因的F<sub>2</sub>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<sub>1</sub>和F<sub>2</sub>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。
A method is described which allows a-human atrial natriuretic factor to be synthesized in stablt form and with high yield in Escherichia coli. Two copies of the synthetic a-hANF gene were linked in tandem, separated by conons specifying a 4-amino-acid linker with glutmic residues flanking the authentic N and C termini of the 28-aa hormone. This linker is for enzymatic cleavage of expressed product by endoproteinase Glu-C. Two copies gene sequence was verified by dideoxynucleotide chain termination method.
出处
《中山医科大学学报》
CSCD
1990年第5期4-7,共4页
Academic Journal of Sun Yat-sen University of Medical Sciences
关键词
心钠素
基因
串联
a-Human atrial natriuretic factor Gene Multiple copies gene
