摘要
目的构建pET30a-EgG1Y162原核表达质粒,对重组蛋白HIS-EgG1Y162进行诱导表达、纯化及活性鉴定。方法从细粒棘球绦虫原头蚴的cDNA中克隆出EgG1Y162基因,构建pMD19-T-EgG1Y162克隆载体。通过EcoRⅠ和HindⅢ双酶切,将EgG1Y162基因片段连接入原核表达载体pET30a中,构建pET30a-EgG1Y162重组质粒,经双酶切及PCR鉴定并测序。将测序正确的重组质粒转化入BL21(DE3)大肠埃希菌的感受态细胞中,利用IPTG诱导表达蛋白,诱导后的菌液经超声破碎,采用12%SDS-PAGE分析蛋白表达情况。使用His Trap纯化柱纯化蛋白,并进行Western blot鉴定。结果PCR扩增出的EgG1Y162基因,片段大小为360 bp,与预期相符。经双酶切及PCR鉴定并测序,成功构建出pET30a-EgG1Y162原核表达载体。重组质粒转化菌经IPTG诱导,HIS-EgG1Y162重组蛋白在菌液上清中的表达量高于沉淀,且在IPTG(0.2 mmol/L)在28℃诱导6 h时在上清中表达量较高。当咪唑浓度为20 mmol/L时重组蛋白的洗脱效果良好。Western blot显示纯化的重组蛋白HIS-EgG1Y162能被相应抗体识别。结论成功构建了pET30a-EgG1Y162重组质粒,并诱导纯化出具有反应原性的重组蛋白HIS-EgG1Y162,为细粒棘球蚴EgG1Y162疫苗的研发奠定了实验基础。
Objective To construct the prokaryotic expression plasmid pET30 a-EgG1 Y162 in order to induce the expression of the recombinant protein His-EgG1 Y162 and to purify and determine the activity of that protein.Methods The gene EgG1 Y162 was cloned from the cDNA of Echinococcus granulosus to construct the plasmid pMD19-T-EgG1 Y162.EcoR I and Hind III were used to ligate EgG1 Y162 gene fragments to the prokaryotic expression vector pET30 a to construct the recombinant plasmid pET30 a-EgG1 Y162.After double enzyme digestion and identification with PCR,the recombinant plasmid was sent for sequencing.The sequence of the recombinant plasmid was verified.The recombinant plasmid was transformed into competent cells(Escherichia coli BL21,DE3),and expression of the protein expression was induced with IPTG.The suspension was sonicated,and protein expression was analyzed using 12%SDS-PAGE.The proteins were purified on a HIS Trap purification column and ultimately identified using Western blotting.Results Amplification with PCR indicated that the EgG1 Y162 fragment was 360 bp in length,which was consistent with expectations.After double enzyme digestion and identification with PCR,sequencing indicated that the prokaryotic expression vector pET30 a-EgG1 Y162 was successfully constructed.Its expression in bacteria transformed with the recombinant plasmid was induced with IPTG.The expression of the recombinant protein His-EgG1 Y162 was higher in the supernatant of suspension than in the precipitate.The level of His-EgG1 Y162 expression in the supernatant was high when it was induced with IPTG(0.2 mmol/L)at 28℃for 6 h.The recombinant protein eluted when 20 mmol/L imidazole was used.The purified recombinant protein His-EgG1 Y162 was ultimately identified using Western blotting.Conclusion The recombinant plasmid pET30 a-EgG1 Y162 was successfully constructed and expression of the recombinant protein His-EgG1 Y162 was induced.The recombinant protein purified and it was found to be reactive.These findings have laid the experimental f
作者
周彦霞
孔慧芳
赵商岐
郑佳
曹春宝
李玉娇
龚巧巧
丁剑冰
周晓涛
ZHOU Yan-xia;KONG Hui-fang;ZHAO Shang-qi;ZHENG Jia;CAO Chun-bao;LI Yu-jiao;GONG Qiao-qiao;DING Jian-bing;ZHOU Xiao-tao(Department of Immunology,College of Basic Medicine,Xinjiang Medical University,Urumqi China 830011;Clinical Laboratory,The First Hospital Affiliated with Xinjiang Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第3期287-291,共5页
Journal of Pathogen Biology
基金
新疆维吾尔自治区自然科学基金项目(No.2018D01C157)。
关键词
质粒构建
重组蛋白HIS-EgG1Y162
原核表达
纯化
鉴定
plasmid construction
recombinant protein HIS-EgG1Y162
prokaryotic expression
purification
identification
