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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。
OBJECTIVE: To construct the prokaryotic expression plasmid pHGhis / c-Aβ-c and express in Escherichia coli to observe the immunogenicity of the fusion protein c-Aβ-c, so as to lay the foundation for the study of genetically engineered vaccine for Alzheimer’s disease (AD) . Methods: Gene fragments of 1 ~ 71,88 ~ 144 amino acids encoding HBcAg (HBc1 ~ 71 and HBc88 ~ 144) and the gene encoding amyloid peptide Aβ1-42 were amplified by PCR. The latter was ligated between HBc1 ~ 71 and HBc88 ~ 144 to construct recombinant plasmid pGEMEX / c-Aβ-c, and the recombinant gene was subcloned into prokaryotic expression vector pHGhis to construct expression plasmid pHGhis / c-Aβ- Induction of expression by temperature. The fusion gene expression was detected by SDS-PAGE (Coomassie brilliant blue staining). BALB / c mice were immunized with fusion protein by intraperitoneal injection. The titer of anti-Aβ antibody in serum of mice was detected by indirect ELISA. Results: The results of DNA sequencing confirmed that the fusion gene was recombined in the expression plasmid, and the expression plasmid was in accordance with the theoretical design. After induction of expression, the expressed protein accounted for about 16% of the bacterial pellet. Anti-Aβ antibody titer in BALB / c mice reached 1:16000 after three immunizations and no anti-HBcAg antibody was detected. CONCLUSION: The c-Aβ-c fusion gene is highly expressed in E. coli and the expressed fusion protein is highly immunogenic.