摘要
【目的】构建Aβ1-15多价DNA疫苗,研究其诱导体液免疫的效果。【方法】基因合成和PCR技术扩增以多个甘氨酸连接的Aβ1-15二价基因片段;在N端引物延伸加上IgGκ轻链信号肽序列;GSGGSGlinker再串连两段Aβ1-15二价基因片段,克隆入pcDNA3.1表达载体,构建pcDNA3.1-s4×Aβ15真核表达质粒;质粒转染COS-7细胞,Westernblot检测4×Aβ15的表达;质粒肌肉注射接种BALB/c鼠,共6次,18周加强免疫,ELISA法检测抗体效价以及抗体分型;免疫组织化学和Aβ42肽抗原的中和实验检测抗血清与转基因鼠Tg2576脑切片中老年斑的特异结合。【结果】测序证实所构建的pcDNA3.1-s4×Aβ15载体序列正确,在COS-7细胞中表达分子质量约8ku的分泌型4×Aβ15蛋白。pcDNA3.1-s4×Aβ15疫苗于第2次加强免疫后产生Aβ抗体,随加强免疫次数增多,抗体滴度增加,在19周,抗体平均滴度为1︰6400,抗体以IgG1型为主,且可以与转基因鼠Tg2576脑切片中的老年斑免疫结合并被Aβ42肽抗原的中和。【结论】所构建的pcDNA3.1-s4×Aβ15疫苗可在小鼠体内产生高效价的Aβ抗体,为下一步免疫老年性痴呆转基因动物的研究提供条件。
[Objective] To construct Aβ1-15 multivalent recombinant DNA vaccine and to investigate the induction of humoral immune response. [Methods] DNA fragment encoding bivalent Aβ1-15 gene was obtained by gene synthesis and PCR technique. The signal peptide of IgG κ light chain was fused to N-terminal of the Aβ15 gene by primer extension. The two fragments of bivalent Aβ15 gene were ligased using GSGGSG gene linkers. The expression vector pcDNA3.1-s4×Aβ15 was constructed. The COS-7 cells were transfected and the expression of 4×Aβ15 were determined by Western blot analysis. BALB/c mice were immunized by intramuscular injection with pcDNA3.1-s4×Aβ15 vaccine, in 18 weeks and detected the titer of antibody and isotyped by ELISA. Mice serum was analyzed to stain Aβ plaque with pcDNA3.1-S4XAβ15 vaccine, in the Tg2576 mouse brain. [Results] DNA sequence identified pcDNA3.1-s4×Aβ15 was constructed correctly, which expressed about 8 ku proteins in COS-7 cell. After six rounds of booster injection, 6 of 8 mice immunized with pcDNA3.1-Aβ42 generated Aβ antibodies that were higher than 1:5000 and the isotype was IgG1. Mice serum bound specially to Aβ plaque in the Tg2576 mouse brain and adding Aβ42 peptide abrogated the reaction. [Conclusion] pcDNA3.1- s4×Aβ15 vaccine generated a significant amount of Aβ antibody, and demonstrated functionally activity of these antibodies and thus showed the potential for therapeutic efficacy.
出处
《中山大学学报(医学科学版)》
CAS
CSCD
北大核心
2005年第4期371-376,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金资助项目(30400512)
广东省自然科学资金重点资助项目(20013137)
广东省自然科学资金资助项目(04300218)
广州市科技计划基金资助项目(2004Z3-E0151)
