Stx1A-LHRH融合毒素基因的構(gòu)建及表達(dá)
發(fā)布時間：2018-10-13 17:32
【摘要】：利用PCR 的方法設(shè)計兩條引物從腸出血性大腸桿菌O157:H7 的基因組DNA 中擴(kuò)增出編碼Stx1A-LHRH 的重組融合基因片段,定向克隆到表達(dá)質(zhì)粒pET28a 中,構(gòu)建重組表達(dá)質(zhì)粒pET28a::Stx1A-LHRH,將重組質(zhì)粒轉(zhuǎn)化到宿主菌BL21(DE3)中。對該重組菌株用IPTG 進(jìn)行誘導(dǎo)表達(dá),并利用超聲波裂解細(xì)菌對表達(dá)蛋白進(jìn)行初步定位。SDS-PAGE 電泳檢測結(jié)果表明重組菌株表達(dá)出了23.7kDa 的目的融合蛋白Stx1A-LHRH,目的蛋白為包涵體表達(dá),經(jīng)薄層掃描分析表明表達(dá)量約占菌體總蛋白的37.6%,約占細(xì)菌超聲波處理后沉淀的63.4%。不同誘導(dǎo)時間的結(jié)果表明,在3-7h 的誘導(dǎo)時間內(nèi),目的蛋白的表達(dá)量沒有明顯變化。利用PCR的方法設(shè)計兩條引物擴(kuò)增出融合基因Stx1A-LHRH,定向克隆到質(zhì)粒pMAL-p2x 中。將重組質(zhì)粒pMAL-p2x:: Stx1A-LHRH 轉(zhuǎn)化到宿主菌TB1 中,對該重組菌株用IPTG 進(jìn)行誘導(dǎo)表達(dá),并利用超聲波裂解處理細(xì)菌。SDS-PAGE 電泳檢測結(jié)果表明重組菌株表達(dá)出了66kDa 的目的融合蛋白MBP-Stx1A-LHRH,且大部分呈可溶性表達(dá)。對表達(dá)出的可溶性蛋白進(jìn)行親和層析純化,純化后的蛋白用factorⅩa 酶切后獲得融合蛋白Stx1A-LHRH。
[Abstract]:Two primers designed by PCR were used to amplify the recombinant fusion gene fragment encoding Stx1A-LHRH from the genomic DNA of E. coli enterohemorrhagic Escherichia coli O157:H7 and cloned into the expression plasmid pET28a. The recombinant expression plasmid pET28a::Stx1A-LHRH, was constructed and transformed into host strain BL21 (DE3). The recombinant strain was induced to express by IPTG, and the expressed protein was preliminarily located by ultrasonic lysis bacteria. The results of SDS-PAGE electrophoresis showed that the recombinant strain expressed the target fusion protein of 23.7kDa, Stx1A-LHRH, which was expressed as inclusion body. Thin-layer scanning analysis showed that the expression amount was about 37.6% of the total bacterial protein, accounting for 63.443% of the precipitation after ultrasonic treatment. The results of different induction time showed that there was no significant change in the expression of the target protein during the 3-7 h induction time. Two primers were designed to amplify the fusion gene Stx1A-LHRH, and cloned into plasmid pMAL-p2x by PCR. The recombinant plasmid pMAL-p2x:: Stx1A-LHRH was transformed into the host strain TB1, and the recombinant strain was induced to express by IPTG. The results of SDS-PAGE electrophoresis showed that the recombinant strain expressed the target fusion protein MBP-Stx1A-LHRH, of 66kDa, most of which was soluble. The expressed soluble protein was purified by affinity chromatography. The purified protein was digested with factor 鈪，
本文編號：2269394
[Abstract]:Two primers designed by PCR were used to amplify the recombinant fusion gene fragment encoding Stx1A-LHRH from the genomic DNA of E. coli enterohemorrhagic Escherichia coli O157:H7 and cloned into the expression plasmid pET28a. The recombinant expression plasmid pET28a::Stx1A-LHRH, was constructed and transformed into host strain BL21 (DE3). The recombinant strain was induced to express by IPTG, and the expressed protein was preliminarily located by ultrasonic lysis bacteria. The results of SDS-PAGE electrophoresis showed that the recombinant strain expressed the target fusion protein of 23.7kDa, Stx1A-LHRH, which was expressed as inclusion body. Thin-layer scanning analysis showed that the expression amount was about 37.6% of the total bacterial protein, accounting for 63.443% of the precipitation after ultrasonic treatment. The results of different induction time showed that there was no significant change in the expression of the target protein during the 3-7 h induction time. Two primers were designed to amplify the fusion gene Stx1A-LHRH, and cloned into plasmid pMAL-p2x by PCR. The recombinant plasmid pMAL-p2x:: Stx1A-LHRH was transformed into the host strain TB1, and the recombinant strain was induced to express by IPTG. The results of SDS-PAGE electrophoresis showed that the recombinant strain expressed the target fusion protein MBP-Stx1A-LHRH, of 66kDa, most of which was soluble. The expressed soluble protein was purified by affinity chromatography. The purified protein was digested with factor 鈪，
本文編號：2269394
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