人纤溶酶原半胱氨酸卷曲区5(hPK-5)基因在4种工程菌株中表达差异性研究

Study on Difference of Expressing Human Plasminogen Kringle 5 (hPK-5) Gene in Four Kinds of Engineering Strains

目的 :探索表达人纤溶酶原半胱氨酸卷曲区 5 (hPK 5 )的最佳菌株 ,为研究其抑制内皮细胞增殖和迁移的活性 ,开发肿瘤治疗和预防肿瘤转移的新药提供前提。方法 :在将人纤溶酶原半胱氨酸卷曲区 5 (hPK 5 )基因与原核表达载体pBV2 2 0进行体外重组获得表达质粒 pBV2 2 0 /hPK 5 ,采用氯化钙转化法将重组质粒分别导入BL2 1(DE3)、DH5α ,JM10 9和BL2 1(DE3) pLyss 4种工程菌 ,在温控诱导表达条件下进行相同表达条件和优化表达条件下的hPK 5因子表达差异性研究。 结果 :工程微生物 (本研究为工程菌株 )的筛选、培养条件的建立、外源基因表达条件的建立是优化基因工程技术体系的重要技术环节。结论

Objective: To explore the difference of expressing the same gene by different engineering strains and to provide the basis of constituting highness expressing system of aim gene.Method: The human plasminogen kringle 5 (hPK 5) gene was amplified a human plasminogen cDNA template by standard polymerase chain reaction(PCR) The amplified cDNA fragment was ligated into the EcoRⅠ and Bam HⅠsites of the pBV220 vector. The hPK 5 plasmid was named pBV220/hPK 5 and then co transfected into E.coli. BL21(DE3), DH5α, JM109 and BL21(DE3) pLyss at the same time. Then the expressing difference was researched in the same condition and the optimizing condition inducing by the controlled temperature.Result: All of these filtrating engineering strains, establishing cultivating condition, and establishing the expression condition of aim gene are three important techniques.Conclusion: The research settle a basic technique for studing human plasminogen kringle 5 and the development of industrialization.