CEA的多表位疫苗基因的设计及构建的表达载体在原核表达系统中的表达  

Designation of CEA Multi-epitope Vaccine and Construction of Expression Vector Expressing in Prokaryotic Expression System

作　　者：王鹏飞[1] 朱茜[1] 张丽芳[2] 朱冠保[1] 

机构地区：[1]温州医学院附属第一医院胃肠外科,325000 [2]温州医学院微生物与免疫教研室

出　　处：《医学研究杂志》2013年第6期76-81,共6页Journal of Medical Research

基　　金：浙江省自然科学基金资助项目(Y2100660)

摘　　要：目的本研究设计肿瘤相关抗原CEA的多表位疫苗基因和构建的重组表达载体在原核表达系统中成功表达融合蛋白。方法根据预测分析得到的表位氨基酸序列,选取预测分值较高的CTL表位(IMIGVLVGV)和B细胞表位(SNNSK-PVEDK),并结合"非选择性"辅助T细胞表位(AKFVAAWTLKAAA)串联起来,按原核系统偏爱的氨基酸密码子优化,委托生物技术公司合成寡核苷酸,退火获得目的基因,并将目的基因克隆至原核表达载体pET32a(+)多克隆位点内。将此重组载体转化E.coli BL21(DE3)表达融合蛋白,并经SDS-PAGE、Western blot分析鉴定。用Ni-NTA树脂亲和层析法纯化表位融合蛋白ozlf-86/87。结果嵌合有多表位目的基因的原核重组质粒pET32a(+)/ozlf-86/87,进行酶切和测序鉴定,构建成功。SDS-PAGE分析表明,重组质粒在37℃1.0mmol/L IPTG条件下诱导6h能很好地表达目的蛋白,相对分子质量(Mr)约为20kDa,与预期Mr大小吻合;蛋白表达量均约占细菌总蛋白量的20%。,并经Western blot鉴定为目的蛋白。用Ni-NTA树脂亲和层析法纯化成功获得表位融合蛋白,即ozlf-86/87,纯度达到90%以上。结论本研究获得的多表位融合蛋白对胃肠道恶性肿瘤诊断试剂的研究和进一步的疫苗研究奠定了基础。Objective Multi - epitope vaccine genes of the tumor - associated antigen CEA in the study was designed and was suc- cessfully constructed a recombinant expression vector of fusion protein expressed in a prokaryotic expression system. Methods Epitope a- mino acid sequence was obtained based on the predictive analysis. The higher prediction score CTL epitopes （IMIGVLVGV） , and B cell epitope （SNNSKPVEDK） were selected, combined with the ＂non -selective＂ helper T cell epitope （AKFVAAWTLKAAA） concatena- ting, original nuclear system favored amino acid codon optimized synthetic oligonucleotides commissioned by the biotechnology company, annealing target gene, and was cloned into the prokaryotic expression vector pET32a （ ＋ ） cloning sites. The recombinant vector was transformed into E. coli BL21 （DE3） to express the fusion proteins, and were identificated by SDS -PAGE and Western Blot analysis. epitope fusion protein ozlf - 86/87 was purified by Ni - NTA resin affinity chromatography. Results Chimeric multi - epitope target gene prokaryotic recombinant plasmid pET32a （ ＋ ） / ozlf - 86/87, was restricted and digested with enzyme and sequenced, and built success- fully. SDS - PAGE analysis showed that the recombinant plasmid well expressed the target protein induced under the conditions of 37 ℃ 1.0mmol/L [PTG for 6h, and that the target protein of 20 kDa in size was expressed; Protein expression approximately accounted for 20% of the total amount of bacteria protein, which was identified as targeted protein by Western blot analysis. The epitope fusion proteins were obtained by using of Ni - NTA affinity resin and chromatography purification method, that is, the purity of ozlf - 86/87 was more than 90%. Conclusion the multiple epitope fusion protein obtained in this study laid the foundation for study of diagnostic reagents for gastro-intestinal cancer and further research of vaccine.

关 键 词：CEA 表位疫苗 质粒 原核表达 蛋白纯化 

分 类 号：R573.1[医药卫生—消化系统]