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【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 polyvalent DNA vaccine and study the effect of inducing humoral immunity. 【Method】 Aβ1-15 bivalent gene fragment with multiple glycines was amplified by gene synthesis and PCR. The signal sequence of IgGκ light chain was amplified by N-terminal primer. The GSGGSGlinker was further linked with two Aβ1-15 bivalent genes The recombinant plasmid pcDNA3.1-s4 × Aβ15 was constructed. The plasmid was transfected into COS-7 cells and the expression of 4 × Aβ15 was detected by Western blot. BALB / c mice were inoculated intramuscularly with 6 The immune titer and antibody typing were detected by ELISA. Immunohistochemistry and neutralization assay of Aβ42 peptide antigen were used to detect the specific binding of antisera to senile plaques in Tg2576 brain slices of transgenic mice. 【Results】 Sequencing confirmed that the constructed pcDNA3.1-s4 × Aβ15 vector sequence was correct, and secreted 4 × Aβ15 protein of about 8 ku in COS-7 cells was expressed. The antibody titer of pcDNA3.1-s4 × Aβ15 after the second booster immunization increased with the number of booster immunization. At 19 weeks, the average antibody titer was 1: 6400, the antibody was mainly IgG1, And immunosorbable to senile plaques in transgenic mouse Tg2576 brain sections and neutralized by A [beta] 42 peptide antigens. 【Conclusion】 The constructed pcDNA3.1-s4 × Aβ15 vaccine can produce high titer Aβ antibodies in mice and provide the conditions for further research on immunization of Alzheimer’s disease-resistant transgenic animals.