人Pcsk9基因原核表达载体的构建及诱导表达.docVIP

人Pcsk9基因原核表达载体的构建及诱导表达 李风梅，俞辉，宋金龙，刘忠民* 广州医学院第一附属医院检验科 广东 广州 510120 The optimization condition for the human pcsk9 recombinant protein in E.coli. LI Feng-mei, YU Hui, SONG Jin-long, LIU Zhong-min* Department of The First Affiliated Hospital OF Guangzhou Medical College, Guangzhou 510120,China [Abstract]: AIM: To construct the expression vector of Proprotein convertase subtilisin/kexin type9 and find the optimum conditions for the target gene expression., prepare for the development of pcsk9 antibody. METHODS: firstly, the sequence of pcsk9 cDNA was synthesized by the biology company; secondly, the sequence was amplified by PCR and cloned into the expression vector PET-28b .After confirmed by DNA sequencing, the recombinant plasmid of pET-28b-pcsk9 was transformed into the competent cell of E.coli.BL21（DE3）.Under different conditions , induce it to express and compare the results. Finally, find the best expression conditions. RESULTS: the objective sequence was cloned and expression in E.coli.BL21（DE3）.The best expression conditions of PET-28b-pcsk9 were at OD=0.6, induced by 1.0 mmol/L IPTG for 8h at temperature of 30℃. CONCLUSION: the expression vector was constructed and expressed well. The expression of the recombinant plasmid of PET-28b-pcsk9 were sensitive to the induced temperature and time. [Key words] : pcsk9 , expression vector, induce expression, optimum condition ［摘要］：目的：构建人前蛋白转化酶枯草溶菌素9的原核表达载体，优化pcsk9诱导表达条件。方法：聚合酶链式反应扩增人工合成的pcsk9 cDNA序列。扩增产物经TA克隆、双酶切、连接，构建pET-28b-pcsk9表达载体；将重组质粒转化入大肠杆菌表达菌BL21（DE3）的感受态细胞中，分别在不同条件下对重组质粒表达菌进行诱导，获得pET-28b-pcsk9的最佳诱导表达条件。结果：成功构建了pET-28b-pcsk9重组表达载体，其最佳表达条件为菌液光密度值取0.6，诱导温度取30℃ ，诱导剂终浓度取1.0 mmol/L，诱导时间取8小时。结论：成功构建了pcsk9原核表达载体pET-28b-pcsk9，该载体在大肠杆菌表达菌BL21（DE3）感受态细胞中可有效地表达，且该重组质粒的表达对诱导所用的温度和时间相对比较敏感。 关键词：pcsk9，表达载体，诱导表达，优化条件 ［中图分类号］：Q78 ［文献标识码］：B ［前言］ 人前蛋白转化酶枯草溶菌素9（Proprotein convertase subt