【摘要】：[目的]構(gòu)建大腸桿菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三種原核表達(dá)系統(tǒng),通過對(duì)比表達(dá)水平篩選出最優(yōu)表達(dá)體系,并純化表達(dá)的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表達(dá)質(zhì)粒構(gòu)建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表達(dá)載體,分別轉(zhuǎn)化大腸桿菌BL21,IPTG誘導(dǎo)表達(dá)Ybf E融合蛋白,對(duì)三種表達(dá)系統(tǒng)的表達(dá)水平進(jìn)行對(duì)比,并對(duì)pET16b-ybf E和pGEX-4T-1-ybf E表達(dá)體系的裂菌上清中的可溶性Ybf E融合蛋白液分別使用鎳柱和GST蛋白純化柱純化。[結(jié)果]構(gòu)建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表達(dá)體系,并使用IPTG誘導(dǎo)表達(dá)Ybf E融合蛋白。ybf E在p GEX-4T-1載體內(nèi)的表達(dá)水平最高,接下來依次為pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表達(dá)的可溶性Ybf E融合蛋白純化后濃度分別為86μg/m L和724μg/m L。[結(jié)論]成功構(gòu)建了ybf E基因的三種原核表達(dá)系統(tǒng),篩選出最佳表達(dá)體系,可溶性Ybf E融合蛋白得到純化。
[Abstract]:[objective] to construct three prokaryotic expression systems of E. coli function unknown gene ybf E, pET16b,pET32a and p GEX-4T-1, and to select the optimal expression system by comparing the expression level, and to purify the expressed soluble Ybf E fusion protein. [methods] the prokaryotic expression vectors of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed by using pET16b,pET32a and p GEX-4T-1 expression plasmids. The expression levels of the three expression systems were compared with those of E. coli BL21,IPTG induced expression of Ybf E fusion protein. The soluble Ybf E fusion protein in the supernatant of pET16b-ybf E and pGEX-4T-1-ybf E expression system was purified by nickel column and GST protein column, respectively. [results] the prokaryotic expression systems of pET16b-ybf pET32a-ybf E and p GEX-4T-1-ybf E were constructed, and the expression level of Ybf E fusion protein. Ybf E was the highest in p GEX-4T-1 vector. The concentration of soluble Ybf E fusion protein expressed by pET16b, pET32a.pET16b-ybf E and p GEX-4T-1-ybf E was 86 渭 g / mL and 724 渭 g / mL, respectively. [conclusion] three prokaryotic expression systems of ybf E gene were successfully constructed, and the best expression system was selected. The soluble Ybf E fusion protein was purified.
【作者單位】： 內(nèi)蒙古大學(xué)生命科學(xué)學(xué)院;內(nèi)蒙古民族大學(xué)生命科學(xué)學(xué)院;
【分類號(hào)】：Q78

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