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.
出处
《中国药学杂志》
EI
CAS
CSCD
北大核心
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 )
