摘要
利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。
The DNA of mouse interleukin 4(MIL-4) gene, consists of 442 base-pairs (bp), was devided into 29 oligonucleotide fragments and synthesized on ABI 381A.DNA synthesizer.The synthetic oligonucleotides were assembled in two groups and inserted into plasmid pUCl2 for cloning.Two recombinant plasmids were formed, in which one plasmid contained the first half gene fragment of MIL-4, the other plasmid contained the second half gene fragment of MIL-4.The two gene fragments were finally identified by DNA sequencing.The total synthetic geae of MIL-4 was obtained by inserting the cloned first half gene of MIL-4 into the recombinant plasmid containing the second half gene of MIL-4 to form recombinant plasmid pFR101.We inserted the total synthetic MIL-4 gene into pSM53 plasmid and transformed E.coli TAP106 According to the effect of MIL-4 to CTLL cell, we confirmed that the active MIL-4 protein was expressed in TAP106(pFR105) cell.
关键词
白细胞介素
基因
合成
克隆
表达
Interleukin 4
Gene chemical synthesis
Gene cloning
Gene expression
