重组基因c-Aβ-c的构建及其表达蛋白的免疫原性分析

目的:构建原核表达质粒pHGhis/c-Aβ-c,并在大肠杆菌中表达,观察融合蛋白c-Aβ-c的免疫原性,为阿尔茨海默病(AD)基因工程疫苗的研究打下基础。方法:采用PCR法,分别扩增编码HBcAg的1~71、88~144氨基酸的基因片断(HBc1~71和HBc88~144),以及编码淀粉样肽Aβ1-42的基因。将后者连接于HBc1~71和HBc88~144之间,构建重组质粒pGEMEX/c-Aβ-c,并将重组基因亚克隆于原核表达载体pHGhis中,构建表达质粒pHGhis/c-Aβ-c,通过温度诱导表达。用SDS-PAGE(考马斯亮蓝染色)检测融合基因的表达。以融合蛋白经腹腔注射免疫BALB/c小鼠,用间接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,融合基因重组于表达质粒之中,表达质粒与理论设计相符。诱导表达后,表达蛋白约占细菌沉淀的16%。BALB/c小鼠经3次免疫后,其血清中抗-Aβ抗体的滴度可达1∶16000,且检测不到抗-HBcAg抗体。结论:c-Aβ-c融合基因在大肠杆菌中可高效表达,表达的融合蛋白具有较强的免疫原性。

Construction and prokaryotic expression of recombinant gene c-Aβ-c and the immunogenicity analysis of the fusion protein

AIM: To construct the recombinant prokaryotic expression plasmid pHGhis/c-Abeta-c and evaluate the immunogenicity of the fusion protein expressed in E.coli DH5 alpha. METHODS: The gene fragments of HBc1-71, HBc88-144 and Abeta(1-42) were amplified by PCR. Then the Abeta(1-42) gene was inserted between HBc1-71 and HBc88-144, yielding the recombinant gene c-Abeta-c. c-Abeta-c gene was cloned into pGEMEX and then subcloned into pHGhis plasmids. c-Abeta-c fusion protein expression in transformed E.coli DH5alpha was induced at 42degrees Celsius. The expressed fusion protein was analyzed by SDS-PAGE. Six BALB/c mice recieved intraperitoneal injection (i.p.) of c-Abeta-c fusion protein purified by saturated ammonium sulfate. The anti-Abeta antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After temperature induction, fusion protein was expressed and existed in the sediment of the bacterial lysate. The expression level was 16% of all the proteins in the sediment. After 3 times of immunization, the titer of anti-Abeta antibody in sera of BALB/c mice reached up to 1:16,000, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-Abeta-c gene can be expressed in E.coli DH5 alpha. The expressed protein exists in the sediment of the bacterial lysate and has a strong immunogenicity. This study lays the foundation for the experimental animal study of Alzheimer's disease genetic engineering vaccine.