水蛭素变异物1活性多肽基因的化学合成及克隆  

CHEMICAL SYNTHESIS AND CLONING OF GENECODING FOR THE ACTIVE POLYPEPTIDEHIRUDIN VARIANT 1

作　　者：郭娟[1] 王荷碧[1] 蔡英林[1] 刘艳彤 顾洁[1] 

机构地区：[1]中国医学科学院血液学研究所

出　　处：《中华血液学杂志》1995年第9期468-470,共3页Chinese Journal of Hematology

基　　金：中国医学科学院科研基金

摘　　要：为了解决天然水蛭素来源匮乏问题，我们采用基因工程技术进行重组水蛭素的研究。根据水蛭素变异物１（ＨＶ１）的氨基酸序列，选择在原核细胞中高表达的密码子，设计了２２１个碱基对长度的ＨＶＩＤＮＡ片段。分１４个寡核苷酸片段进行化学合成，各片段连接成ＨＶ１全基因后，经ＥｃｏＲＩ、ＢａｍＨＩ切点克隆入ｐＵＣ１８／１９质粒，转化大肠杆菌ＪＭ１０５，蓝白菌落法筛选阳性克隆。限制性内切酶实验及ＤＮＡ序列分析证实了ＨＶ１全基因的正确性，克隆成功。并且获得活性表达。ased on the amino acid sequence of hirudin variant1 (HV 1), we designed a 221bp HV 1 DNA fragmentcontaining codons normally coding for highly expressedin E. coli proteins. The whole DNA fragment was ob-tained by ligation of 14 individual short oligonu-cleotides fragments (HF1～HF14) synthesized chemi-cally , and was inserted into the EcoRI/BamHI restric-tion sites of the PUC 18/19 vector. The ligation mix-ture was used to transform E. coli JM105. Clones werescreened by IPTG/X-Gal assay. Restriction enzymicassay and DNA sequence analysis of the positive clonesconfirmed the existence of the designed gene.

关 键 词：水蛭素变异物1 重组 化学合成 

分 类 号：R394.8[医药卫生—医学遗传学] R973.2[医药卫生—基础医学]