携带tPA信号肽序列HuZP3核心抗原核酸疫苗质粒的构建与体外瞬时表达  

Construction and transient expression of HuZP3 core antigen fused to tPA signal sequence encoded by nucleic acid vaccine plasmid in vitro

作　　者：郭焱[1] 瞿新明[1] 崔健丽[2] 吴泽民[1] 李春莉[3] 金宁一[4] 

机构地区：[1]长春中医药大学基础医学院,长春130117 [2]吉林大学第四医院,长春130021 [3]吉林省人民医院,长春130012 [4]军事医学科学院军事兽医研究所,长春1301121

出　　处：《中国免疫学杂志》2014年第1期73-76,共4页Chinese Journal of Immunology

基　　金：吉林省科技厅计划发展项目(20140204001YY)

摘　　要：目的:构建含有组织型纤溶酶原激活剂(tPA)信号肽的人卵透明带3(HuZP3)蛋白核心抗原核酸疫苗质粒并进行体外瞬时表达。方法:将HuZP3基因用PCR方法扩增并分别引入限制性内切酶酶切位点,然后克隆入pIRESIneo载体中获得核酸疫苗质粒,经筛选鉴定后,将上述核酸疫苗质粒及空载体质粒pIRESIneo分别用脂质体瞬时转染CHO细胞,应用免疫印迹法检测核心抗原在体外CHO细胞中的表达,并将其免疫实验小鼠,以酶联免疫吸附法验证其免疫原性。结果:成功构建以pIRESIneo为载体的含tPA信号肽的HuZP3核心抗原核酸疫苗质粒,体外瞬时表达证实含tPA信号肽的HuZP3核心抗原在CHO细胞胞内和胞外均能表达,含tPA信号肽的HuZP3核心抗原核酸疫苗质粒的核心抗原表达水平较高,核酸疫苗质粒具有很好的免疫原性。结论:以pIRESIneo为载体的含tPA信号肽的HuZP3核心抗原核酸疫苗能够将细胞内的HuZP3核心抗原分泌到细胞外,构建的核酸疫苗质粒pIRES1-ZP3/tPA具有很好的免疫原性,为进一步研究其免疫原性奠定了基础。Objective： To construct and transiently express nucleic acid vaccine plasmid encoding human zona pellucida 3 （HuZP3） protein fused to tissue plasminogen activator signal sequence（tPA） in vitro. Methods： Restriction sites were introduced to HuZP3 gene using polymerase chain reaction（PCR）. The gene product was cloned to pIRESIneo to construct nucleic acid vaccine plasmid and all constructs were identified by sequencing, and CHO cells were transiently transfected with pIRESIneo/ HuZP3 /tPA and plRESIneo blank vector. The HuZP3 protein expressed in the CHO cells was measured by Western blot,we immuned the mice by it and verified its immunogenicity by enzyme-linked immunosorbent method. Results： pIRESIneo/HuZP3/tPA nucleic acid vaccine plasmid was constructed successfully and transiently expressed HuZP3 core antigen in intracellular and extracellular lysates and supernatants in CHO cells in vitro. Expression level of pIRESIneo/HuZP3/tPA was higher than that of pIRESIneo/HuZP3. The nucleic acid vaccine plasmid had good immunogenicity. Conclusion： HuZP3 core antigen encoded by nucleic acid vaccine pIRESIneo/HuZP3/tPA and the expressed product in CHO cells could be secreted out of the cells. The nucleic acid vaccine plasmid pIRES1 ZP3 / tPA we built has good immunogenicity, this research is the foundation for the further study of the imnmnogenicity of the pIRESIneo/HuZP3/tPA nucleic acid vaccine.

关 键 词：HuZP3核心抗原 核酸疫苗 瞬时表达 免疫原性 

分 类 号：R512.62[医药卫生—内科学]