蚯蚓溶栓酶基因的克隆及非融合性表达  

Cloning and inclusion bodies expression of the gene of earthworm fibrin olytic enzyme

作　　者：陈飞[1] 李莉[1] 孟宪志[1] 梁国栋[2] 刘艳玲[1] 吴红艳[1] 

机构地区：[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

出　　处：《中国药学杂志》2003年第12期961-963,共3页Chinese Pharmaceutical Journal

基　　金：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 (Lumbricusbimastus)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 pBV2 2 0 P3 0 重组质粒 ,在大肠杆菌DH5α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 888bp ,编码含 2 4 2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P3 0 ,经活性测定 ,具有纤溶活性。结论　P3 0 为首次发现的新基因 ,在国际基因库的输入号为GenebankAF 10 96 4 8,并获得国家专利 ,专利号为 9810 2 2 5 7.X。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 expression 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 × 103 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.

关 键 词：蚯蚓溶栓酶 基因克隆 非融合性表达 双胸蚓属 重组表达 

分 类 号：R977.3[医药卫生—药品] R96[医药卫生—药学]