融合蛋白d-EGF的结构预测、克

发布时间：2019-01-30 12:24

【摘要】：目的：对融合蛋白d-EGF二级、三级结构进行预测并分析其活性部位的可能影响，进行原核表达，并对表达产物进行分析鉴定。方法：以融合蛋白质基因序列为基础，用DNAStar软件预测其蛋白质的二级结构；用Insight II软件对其三级结构进行建模预测。在对融合蛋白理论预测的基础上，分别构建含β-defensin-3、EGF的重组质粒，应用重组PCR等方法，将EGF的基因序列融合到β-defensin-3DNA序列的3’末端，将融合基因克隆入原核表达载体pET-SUMO,构建目的重组质粒pET-SUMO-d-EGF。采用PCR、测序等方法对目的基因是否正确重组进行鉴定；以重组质粒转化大肠杆菌E.coli BL21（DE3），以IPTG诱导表达目的蛋白质，，表达产物经过Ni-trap柱纯化，SDS-PAGE检测表达初产物及纯化产物。最后用Western-blot对其特异性进行鉴定；用MTT法检测其促细胞增殖活性；用纸片扩散法药物敏感试验、微量稀释法检测其抗菌活性。结果：采用DNAStar软件的Gamier Robson方法预测的结果表明，融合蛋白质d-EGF含较多的β折叠结构；Charge Density-Charge法预测蛋白质3-22、31-48区段带丰富的正电荷；Insight II软件分析显示重组蛋白d-EGF中原蛋白β-defensin-3、EGF三级结构中的活性必须的六个链内二硫键在融合蛋白中都能够正确形成，维持β-defensin-3三级结构的3个反向平行的β折叠片结构在融合蛋白中能够形成。融合蛋白的结构含有原蛋白β-defensin-3、EGF的生物活性结构。成功构建β-defensin-3、EGF重组质粒，经重组PCR成功扩增出294bp的目的片段，重组体PCR结果与预期结果一致，测序正确。转入重组质粒的E.coli BL21（DE3）经IPTG诱导、SDS-PAGE分析得到28kD左右的目的蛋白条带。用Western-blot鉴定出融合蛋白含有EGF，β-防御素-3的相应蛋白抗原表位。用MTT法测得融合蛋白d-EGF EC50约为0.4ng/ml,与EGF标准品EC500.08-0.8ng/ml相符；用纸片扩散法药物敏感实验检测对金黄色葡萄球菌有明显抑菌环出现，抑菌浓度25μg/ml相对标准品h β-defensin-3的MIC12.5μg/ml，说明d-EGF抑菌活性比标准品相对弱一些。结论：在预测融合蛋白d-EGF的二、三级结构的基础上，成功构建原核表达载体pET-SUMO d-EGF,并表达出纯化的融合蛋白，且融合蛋白d-EGF具有促细胞增殖和抗菌活性。
[Abstract]:Aim: to predict the secondary and tertiary structure of the fusion protein d-EGF and analyze the possible effects of its active site on prokaryotic expression. Methods: based on the fusion protein gene sequence, the secondary structure of its protein was predicted by DNAStar software, and its tertiary structure was modeled and predicted by Insight II software. Based on the prediction of fusion protein theory, the recombinant plasmids containing 尾-defensin-3,EGF were constructed, and the gene sequence of EGF was fused to the 3 'terminal of 尾-defensin-3DNA by means of recombinant PCR. Construction of Recombinant plasmid pET-SUMO-d-EGF. by cloning Fusion Gene into prokaryotic expression Vector pET-SUMO, PCR, sequencing was used to identify the correct recombination of the target gene. The recombinant plasmid was transformed into Escherichia coli E.coli BL21 (DE3), and the target protein was induced by IPTG. The expressed product was purified by Ni-trap column, and the expressed product and purified product were detected by SDS-PAGE. Finally, its specificity was identified by Western-blot, the cell proliferation activity was detected by MTT assay, the drug sensitivity test by disk diffusion method and the antimicrobial activity by microdilution method. Results: the results of Gamier Robson method using DNAStar software showed that the fusion protein d-EGF contained more 尾 -fold structure, and the Charge Density-Charge method was used to predict the rich positive charge in protein 3-22N 31-48 region. The analysis of Insight II software showed that the disulfide bonds in the six chains necessary for the activity of the proto-protein 尾-defensin-3,EGF in the recombinant protein d-EGF could be formed correctly in the fusion protein. Three reverse parallel 尾-fold structures that maintain the tertiary structure of 尾-defensin-3 can be formed in the fusion protein. The structure of the fusion protein contains the bioactive structure of proto-protein 尾-defensin-3,EGF. The recombinant plasmid of 尾-defensin-3,EGF was successfully constructed and the target fragment of 294bp was successfully amplified by recombinant PCR. The result of PCR was consistent with the expected result and the sequencing was correct. E.coli BL21 (DE3) transformed into the recombinant plasmid was induced by IPTG, and the target protein bands about 28kD were obtained by SDS-PAGE analysis. The corresponding protein epitopes containing EGF, 尾-defensin-3 were identified by Western-blot. The fusion protein d-EGF EC50 measured by MTT method was about 0.4 ng / ml, which was consistent with EGF standard EC500.08-0.8ng/ml. The antimicrobial activity of d-EGF was weaker than that of standard h 尾-defensin-3, and the inhibitory concentration of 25 渭 g/ml was relative to MIC12.5 渭 g / ml of h 尾-defensin-3. Conclusion: on the basis of predicting the secondary and tertiary structure of d-EGF, the prokaryotic expression vector pET-SUMO d-EGF was successfully constructed, and the purified fusion protein was expressed. The fusion protein d-EGF has the ability of promoting cell proliferation and antibacterial activity.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

【相似文献】