Stx1A-LHRH融合毒素基因的构建及表达
发布时间：2018-10-13 17:32
【摘要】：利用PCR 的方法设计两条引物从肠出血性大肠杆菌O157:H7 的基因组DNA 中扩增出编码Stx1A-LHRH 的重组融合基因片段,定向克隆到表达质粒pET28a 中,构建重组表达质粒pET28a::Stx1A-LHRH,将重组质粒转化到宿主菌BL21(DE3)中。对该重组菌株用IPTG 进行诱导表达,并利用超声波裂解细菌对表达蛋白进行初步定位。SDS-PAGE 电泳检测结果表明重组菌株表达出了23.7kDa 的目的融合蛋白Stx1A-LHRH,目的蛋白为包涵体表达,经薄层扫描分析表明表达量约占菌体总蛋白的37.6%,约占细菌超声波处理后沉淀的63.4%。不同诱导时间的结果表明,在3-7h 的诱导时间内,目的蛋白的表达量没有明显变化。利用PCR的方法设计两条引物扩增出融合基因Stx1A-LHRH,定向克隆到质粒pMAL-p2x 中。将重组质粒pMAL-p2x:: Stx1A-LHRH 转化到宿主菌TB1 中,对该重组菌株用IPTG 进行诱导表达,并利用超声波裂解处理细菌。SDS-PAGE 电泳检测结果表明重组菌株表达出了66kDa 的目的融合蛋白MBP-Stx1A-LHRH,且大部分呈可溶性表达。对表达出的可溶性蛋白进行亲和层析纯化,纯化后的蛋白用factorⅩa 酶切后获得融合蛋白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，
本文编号：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，
本文编号：2269394
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