防御素基因的化学合成及克隆

CHEMICAL SYNTHESIS AND CLONING OF DEFENSIN GENE

将防御素基因分成４个寡聚核苷酸片段，采用固相亚磷酰胺法在ＤＮＡ合成仪上化学合成。片段２和４分别用Ｔ＿４多聚核苷酸激酶将５′端磷酸化，４个片段等量混合，用Ｔ＿４ＤＮＡ连接酶连成一个１０９ｂｐ的双股ＤＮＡ片段，其两侧为ＢａｍＨＩ和ＳａｌⅠ粘性末端。把它与经上述双酶切的质粒ｐＵＣ１９连接，转化大肠杆菌ＤＨＳα。转化株经含Ｘｇａｌ青霉素平板初筛，质粒酶切分析以及ＰＣＲ专一性扩增，证实已获得完整的防御素基因克隆。为进一步表达防御素提供了材料．

Four oligonueleotides of delinsin gene were chemically synthesized by the solid-phase phosphoramidite method.Fragment 2 and 4 were phos phorylated at the 5'end ,and all of four fragments were ligased to form a 109 bp DNA duplex.It was ligasea with plasmid pUC 19 and the recombinant plasmid was transformed into E.coli DH5α.By means of Xgal plate,electrophoresis and ploymerase chain reacton,it is verified that the defensin gene was cloned.