【摘要】 目的:原核表达并纯化含β-淀粉样肽(β-amyloid peptide,Aβ)氨基段15肽(Aβ1–15)和删除c/e1表位的截断型HBcAg的融合蛋白,观察其形成的病毒样颗粒,检测其免疫原性,为阿尔茨海默病(Alzheimer’sdisease,AD)基因工程疫苗的研究提供基础。方法:将合成的Aβ1–15基因连接于HBcAg的1~71的3′端,再将HBcAg的88~144位氨基酸的基因片断连接于Aβ1-15的基因的3′端,构建重组质粒pUC/c-Aβ15-c,将重组基因亚克隆于原核表达载体pET-28a(+)中,构建表达质粒pET/c-Aβ15-c。异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导、SDS-PAGE和考马斯亮蓝染色检测重组基因的表达。表达的融合蛋白(命名为CA15C)纯化后,透射电镜观察病毒样颗粒的形成。以病毒颗粒抗原CA15C腹腔注射免疫昆明小鼠,间接ELISA法检测小鼠血清中抗-Aβ抗体的滴度。结果:经酶切鉴定、DNA序列测定证实,目的基因重组于表达质粒之中,与理论设计相符。诱导表达后,在细菌裂解液的上清和沉淀中均可见表达蛋白CA15C,以沉淀中为多,约占细菌沉淀总蛋白的40%。电镜下可见纯化后的CA15C形成直径约30 nm的病毒样颗粒。昆明小鼠经CA15C免疫5次后,其血清中抗-Aβ抗体的滴度可达1:10000,且检测不到抗-HBc抗体。结论:原核表达制备的含Aβ1-15和HBcAg的融合蛋白CA15C,可形成病毒样颗粒,有较强的免疫原性。更多还原

【Abstract】 Objective: To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ15-c,and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli.Methods: The gene fragment HBc88–144was ampli ed by PCR and subcloned to pUC19.The synthetic,double-strand Aβ1-15gene was inserted downstream of HBc1–71in pGEMEX/c1–71.After restrictionenzymedigestion,c1–71-Aβ15wassplicedtoHBc88–144,yieldingtherecombinantgenec-Aβ15-c;that gene was subcloned into pET-28a(+).The fusion protein(CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside(IPTG) and analyzed by SDS-PAGE.The virus-like particle(VLP) formed by fusion protein CA15C was observed with transmission electric microscope(TEM).Four Kunming(KM) mice were given intraperitoneal injections of CA15C,and the anti-Aβ antibody elicited was detected by indirect ELISA.Results:The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing.After IPTG induction,the fusion protein was expressed,mainly in the sediment from the bacterial lysate.The expression level was 40% of total protein in the sediment.The CA15C could form VLP.After 5 rounds of immunization,the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000,while the anti-HBc antibody was undetectable.Conclusion: Recombinant c-Aβ 15-c gene can be expressed in E.coli.The expressed protein can form VLPs and has a strong immunogenicity.更多还原