间日疟原虫多表位疫苗基因的表达、纯化与初步鉴定

Expression,purification and initial identification of the multi-epitope gene of Plasmodium vivax in Pichia pastoris

目的构建和筛选间日疟原虫多表位疫苗基因高拷贝Pichia pastoris菌株,研究多表位疫苗基因在酵母菌中的表达,纯化目的产物,为进一步的多表位肽免疫原性研究奠定基础。方法根据文献报道筛选间日疟原虫有效保护性抗原表位,人工合成多表位基因PvDBW,经EcoRⅠ和NotⅠ双酶切构建酵母表达载体pPIC9K-PvDBW,转化大肠埃希菌ToP10;鉴定后转化P.pastoris GS115,构建酵母表达菌株;通过G418压力筛选筛出目的基因高拷贝克隆,用甲醇诱导多表位肽基因表达,分别以RT-PCR和Western blot进行鉴定,用50%饱和硫酸铵沉淀和分子筛凝胶层析分离、纯化目的产物。结果重组质粒用EcoRⅠ和NotⅠ双酶切后可得到786 bp DNA片段;经G418压力筛选筛出两个高拷贝菌株,以α-Factor和3′AOX1为引物、重组菌基因组DNA为模板,PCR扩增出约960 bp的目的片段;SDS-PAGE显示,在GS115细胞内多表位肽呈分泌性表达,表达量为80 mg/L;分离、纯化后,纯度达90%以上;Western blot检测显示表达的多表位肽可被间日疟病人血清识别。结论间日疟原虫多期多阶段多表位疫苗基因在酵母菌中表达成功。

Objective To express the multi epitope gene（PvDBW） of Plasmodium vivax in Pichia pastoris. Methods A multi epitope gene（PvDBW） of P. vivax was designed, synthesized, cloned into expression vector pPICgK, and expressed in P. pastoris GS115 by induced with 1% methanol. The expressed product was purified, identified with RT- PCR and Western blot. Results A 786 bp multi-epitope gene（PvDBW） of P. vivax was designed and synthesized, and recombinant plasmid pPIC9K/PvDBW was constructed. SDS-PAGE showed that the gene was expressed in form of soluble molecule in supernatant of induced pPIC9K/PvDBW/GS115 and the rate of the interesting peptide was 80 mg/L. The peptide was specifically recognized by patients＇ sera infected with P. vivax. The purity of expressed product reached above 90% after purified. Conclusion Recombinant plasmid pPICgK/PvDBW is successfully constructed and the multiepitope peptide of P. vivax is expressed in P. pastoris in supernatant in the form of soluble molecule. These results set up a foundation for the further experiments in animals.