eglinC基因的化学合成与克隆

CHEMICAL SYNTHESIS AND CLONING EGLIN C GENE

采用固相亚磷酰胺法合成了ｅｇｌｉｎＣ全基因，该基因全长２２１ｂｐ，包括其结构基因，５＇－端起始密码子ＡＴＧ和３＇－端终止密码子ＴＡＧ，并在基因的５＇－和３＇－分别装上ＥｃｏＲＩ和ＢａｍＨＩ位点．共分１２个片段，采取分段合成，酶促连接成完整的ｅｇｌｉｎＣ基因．合成的基因克隆于Ｍ１３载体，经杂交、检测，筛选出含ｅｇｌｉｎＣ基因的克隆．用双脱氧链终止法测其序列，结果与设计的一致．

A total gene for eglin C has been chemically synthesized by using the solid phosphoramidite method. The gene is 221 bp in length containing the structure gene encoding 70 amino acids,a start coden ATG at 5'-end and a stop coden TAG at 3'-end of the structure gene,except two restriction sites at the ends. The synthesiS of the gene involved enzymatic joining synthetic oligonucleotiede fragments to form DNA duplexes, cloning into vector M13, idnetifying by dot bloting. Finally,the synthetic coding sequence was fully verified right by dideoxynucleotide chain termination method.