人血管生长素基因的化学合成与克隆

采用固相亚磷酸胺法化学合成并克隆人血管生长素基因，该基因全长４２３ｂｐ，其中３６９ｂｐ的结构基因全部选用大肠杆菌偏爱的密码子，在结构基因上游设置了适合原核非融合蛋白表达的核糖体结合位点的Ｓ－Ｄ序列。采用两级退火一步拼接法成功克隆全长基因，该基因适合在多种融合或非融合原核表达载体中表达。

Chemical synthesis and clone of human angiogenin gene

423-bp long Angiogenin(ANG) gene was divided into 20 28-46 mers DNA fragments and chemically synthesized on ABI 381A DNAsynthesiser seperately. Twostep annealling and ligation were performed to assemble the gene. Then the gene was cloned into M13mp18 vector and sequenced on ABI 373A DNA sequencer using Dye Primer Cycle Sequencing Kit. The 369-bp Angiogenin structural gene corresponding to the mature portion of the protein was designed to use preferred E. colt codons according to the amino acid sequence of human angiogenin. Upstream from the structural gene,theCll ribosome binding site (RBS), start codon ATG and KpnI, Hpal sites were added.The stop codon TGA,TAA were located immediately downstream from the structuralgene followed by Hpal and BamHI sites. The sequence of the elf RBS was modified to create an EcoRI site immediately upstream from the ATG. The gene has been highly efficiently expressed in PWR450-1 and PBV220 prokaryotic expression vectors as the fusion protein and intact protein,respectively.