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人睾丸前列腺素D合成酶重组表达质粒的构建与鉴定 被引量:5

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摘要 目的 :构建高效表达人睾丸前列腺素D合成酶 (htL PGDS)的重组表达质粒。 方法 :以经测序证实为人L PGDS的人睾丸L PGDScDNA为模板进行PCR扩增 ,得到编码L PGDS的基因片段 ,用限制性内切酶BamHⅠ和EcoRⅠ进行酶切后 ,连接到经相同内切酶处理的原核表达载体pGEX 2T中。重组质粒pGEX 2T/htL PGDS被进一步转化感受态大肠杆菌JM10 3 ,转化物接种到含氨苄青霉素的LB平板 ,37℃培养 12~ 16h后 ,挑取氨苄青霉素抗性菌落 ,并接种到含氨苄青霉素的LB液体培养基中 ,37℃、16 0r/min培养 12~ 16h后 ,提取质粒DNA供PCR和双酶切鉴定。 结果 :共筛选出氨苄青霉素抗性菌落 10 9个 ,经PCR和双酶切鉴定出 10个重组质粒pGEX 2T/htL PGDS。结论 :重组表达质粒pGEX 2T/htL PGDS的构建及正确鉴定 ,为以后的重组抗原的表达、纯化及单克隆抗体的制备奠定了基础。
出处 《中华男科学杂志》 CAS CSCD 2000年第4期230-233,共4页 National Journal of Andrology
基金 解放军九五计划生育基金!资助项目 (编号 :军计生 98 0 4号 )
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