發(fā)布時(shí)間：2018-09-14 13:16

【摘要】：目的構(gòu)建人前蛋白轉(zhuǎn)化酶枯草溶菌素9(pcsk9)的原核表達(dá)載體,優(yōu)化pcsk9誘導(dǎo)表達(dá)條件。方法聚合酶鏈?zhǔn)椒磻?yīng)擴(kuò)增人工合成的pcsk9 cDNA序列。擴(kuò)增產(chǎn)物經(jīng)雙酶切、連接,構(gòu)建pET-28b-pcsk9表達(dá)載體;將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌表達(dá)菌BL21(DE3)的感受態(tài)細(xì)胞中,分別在不同條件下對(duì)其進(jìn)行誘導(dǎo),獲得pET-28b-pcsk9的最佳誘導(dǎo)表達(dá)條件。結(jié)果成功構(gòu)建了pET-28b-pcsk9重組表達(dá)載體,其最佳表達(dá)條件為菌液光密度值取0.6,誘導(dǎo)溫度取30℃,誘導(dǎo)劑終濃度取1.0 mmol/L,誘導(dǎo)時(shí)間取8 h。結(jié)論該重組表達(dá)載體構(gòu)建成功,在大腸桿菌表達(dá)菌BL21(DE3)中可有效地表達(dá),對(duì)誘導(dǎo)所用的溫度和時(shí)間相對(duì)比較敏感。
[Abstract]:Objective to construct the prokaryotic expression vector of human proprotein converting enzyme lysogenin 9 (pcsk9) and to optimize the pcsk9 induced expression conditions. Methods the synthetic pcsk9 cDNA sequence was amplified by polymerase chain reaction. The amplified product was digested by double enzyme and ligated to construct the pET-28b-pcsk9 expression vector. The recombinant plasmid was transformed into the competent cells of Escherichia coli expression strain BL21 (DE3) and induced under different conditions to obtain the best induced expression conditions of pET-28b-pcsk9. Results the recombinant expression vector of pET-28b-pcsk9 was successfully constructed. The optimal expression conditions were as follows: the optical density of the liquid was 0.6, the induction temperature was 30 鈩，

本文編號(hào)：2242817