多房棘球绦虫多表位疫苗GILE的设计构建及原核表达

Construction and prokaryotic expression of multiepitope vaccine GILE of Echinococcus multilocularis ZHOU Pei;,XIN Mingyuan;,MA Jingwei;,HU Binwen;,WANG Lei

目的 设计、构建多房棘球绦虫多表位疫苗GILE,并表达、纯化该重组蛋白。方法 基于课题组前期对多房棘球绦虫EmEMY162、EmLAP、EmGLUT1优势抗原表位鉴定的结果,通过SWISS-MODEL、pyMOL、SOPMA、VMD生物信息学软件预测GILE的结构及疏水性,设计多表位疫苗GILE。合成GILE序列,插入质粒pCzn1中,构建重组质粒pCzn1-GILE。将重组质粒转化入大肠杆菌Artic express中,添加IPTG诱导重组蛋白表达。通过SDS-PAGE和免疫印迹法确定重组蛋白是否表达。结果 通过SWISS-MODEL软件开展同源建模,结果表明抗原表位最优串联方式为EMY162_(95-104)―LAP_(464-479)―LAP_(495-510)―LAP_(396-410)―EMY162_(106-121)―LAP_(504-518)―EMY162_(112-126)。通过SOPMA软件分析GILE的二级结构,结果表明α螺旋占13.84%,β折叠占26.25%,β转角占14.88%,无规卷曲占45.82%。通过酶切和测序验证表明,质粒pCzn1-GILE构建成功。通过IPTG诱导,得到45KD的重组蛋白。通过Ni-NTA柱的亲和纯化,得到纯化重组蛋白GILE。通过Western blotting法验证,纯化后的GILE蛋白能与His抗体特异结合,得到纯化重组蛋白GILE。结论 本研究通过生物信息学软件成功设计、构建了多表位疫苗GILE,并通过GILE原核表达系统表达、纯化了重组蛋白GILE。

Objective To design and construct the multiepitope vaccine GILE of Echinococcus multilocularis as well as express and purify the recombinant protein.Methods Based on the identification results of dominant epitopes of Echinococcus multilocularis EmEMY162,EmLAP and EmGLUT1 in the early stage of the research group, the structure and hydrophobicity of GILE were predicted by bioinformatics software including SWISS-MODEL,pyMOL,SOPMA and VMD,and the multiepitope vaccine GILE was designed.GILE sequence was synthesized and inserted into plasmid pCzn1 to construct recombinant plasmid pCzn1-GILE.The recombinant plasmid was transformed into E.coli Artistic express and IPTG was added to induce the expression of the recombinant protein.Results The SWISS-MODEL homology modeling showed that the optimal connection mode was EMY162_(95-104)―LAP_(464-479)―LAP_(495-510)―LAP_(396-410)―EMY162_(106-121)―LAP_(504-518)―EMY162_(112-126).The secondary structure of GILE was Alpha helix(14.88%),Extended strand(26.25%),Beta turn(3.73%)and Random coil(45.82%)in GILE.The restrict enzyme digestion and sequencing data showed that the plasmid pCzn1-GILE was successfully constructed.45 KD recombinant protein was induced by IPTG.The purified recombinant protein GILE was obtained by affinity purification on Ni-NTA column.Western Blot showed that the purified GILE protein could specifically bind to His antibody to obtain the purified recombinant protein GILE.Conclusion The multiepitope vaccine GILE is successfully designed and constructed by bioinformatics software, and the recombinant protein GILE is expressed and purified by GILE prokaryotic expression system.