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蚯蚓溶栓酶基因的克隆及非融合性表达
引用本文:陈飞,李莉,孟宪志,梁国栋,刘艳玲,吴红艳.蚯蚓溶栓酶基因的克隆及非融合性表达[J].中国药学杂志,2003,38(12):961-963.
作者姓名:陈飞  李莉  孟宪志  梁国栋  刘艳玲  吴红艳
作者单位:1. 辽宁省微生物科学研究院,辽宁,朝阳,122000
2. 中国预防医学科学院病毒研究所,北京,100052
基金项目:国家高新技术研究发展计划 ( 863 )生物技术资助项目 ( 10 2 -0 8-0 2 -0 3 )
摘    要: 目的克隆和表达我国特有的双胸蚓属蚯蚓(Lumbricus bimastus)体内一种具有纤溶活性的蛋白质。方法通过蛋白质N-末端测序、引物设计及合成、RT-PCR得其对应的cDNA,随后构建了pBV220-P30重组质粒,在大肠杆菌DH中进行非融合性表达,再经亲和色谱柱纯化复性,得到重组蚯蚓溶栓酶蛋白。结果 克隆cDNA全长888 bp,编码含242个氨基酸的成熟肽,SDS-PAGE电泳显示分子量在30×10-3左右,因此将其命名为P30,经活性测定,具有纤溶活性。结论P30为首次发现的新基因,在国际基因库的输入号为Genebank AF-109648,并获得国家专利,专利号为98102257.X。

关 键 词:双胸蚓属  蚯蚓纤溶酶  基因克隆  重组表达
文章编号:1001-2494(2003)12-0961-03
收稿时间:2003-01-17;
修稿时间:2003年1月17日

Cloning and inclusion bodies expression of the gene of earthworm fibrinolytic enzyme
CHEN Fei ,LI Li ,MENG Xian-zhi ,LIANG Guo-dong ,LI U Yan-ling ,WU Hong-yan.Cloning and inclusion bodies expression of the gene of earthworm fibrinolytic enzyme[J].Chinese Pharmaceutical Journal,2003,38(12):961-963.
Authors:CHEN Fei  LI Li  MENG Xian-zhi  LIANG Guo-dong  LI U Yan-ling  WU Hong-yan
Institution:CHEN Fei 1,LI Li 1,MENG Xian-zhi 1,LIANG Guo-dong 2,LI U Yan-ling 1,WU Hong-yan 1
Abstract:OBJECTIVE To study the cloning and inclusion bodies expression of one kind of proteins from earthworm (Lumbricus Bimastus)in China. METHODS N-terminal of protein was sequenced.The PCR primer was designed according to the N-terminal sequenced.Its cDNA fragment was obtained by PT-PCR,then it was expressed in pBV220-P30expression system as inclusion bodies protein.The protein was purified by Sepharose 4B to achieve the protein of earthworm fibrinolytic enzyme. RESULTS The sequence showed that the fragment was 888 bp,with ORF of 726 bp and the 3'untranslation terminal area.The ORF encoded a protein of 242 amino acids.The molecular mass about 30×10-3 was extracted by SDS-PAGE.It was named as P30.The protein had fibronolytic activity. CONCLUSION A new gene was found for the first time and Genbank password is Genbank AF 109648.It has achieved the national patent with the patent mumber 98102257.X.
Keywords:Lumbricus bimaslus    fibrinolytic enzyme  clonin g  recombination and expression
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