【摘要】 目的:构建β-淀粉样蛋白(Aβ42)的多价重组DNA疫苗,确定是否诱导较高滴度Aβ抗体的产生.方法:PCR法从Tg2576转基因鼠基因组DNA中扩增人Aβ42基因片段;在Aβ42基因的5′端引物延伸连接IgGк轻链信号肽序列;利用GSGGSGlinker基因串连两段Aβ42基因片段;将产物克隆入pcDNA3.1表达载体,分别构建pcDNA3.1Aβ42,一价分泌型pcDNA3.1-sAβ42,二价分泌型pcDNA3.1-s2×Aβ42载体;转染COS-7细胞,免疫印迹检测Aβ42的表达;im接种BALB/c鼠,ELISA法测定抗体效价;免疫组织化学和Aβ42肽抗原的中和实验检测抗体与Tg2576鼠脑切片中的老年斑的结合.结果:测序证实所构建的pcDNA3.1-Aβ42,pcDNA3.1s-Aβ42,pcDNA3.1-s2×Aβ42载体插入序列正确;WesternBlotting证实在COS-7细胞中表达相应的蛋白.经6轮,22wk加强免疫后,pcDNA3.1-Aβ42疫苗不能产生Aβ抗体、pcDNA3.1sA-β42接种后产生较低的Aβ抗体(<1∶1000),pcDNA3.12×Aβ42第1次加强免疫后即产生抗体,抗体滴度随接种次数增多而升高,22wk时抗体平均滴度达1∶5000以上,可以与转基因鼠Tg2576脑切片中的老年斑结合,并能被Aβ42肽抗原特异性中和.结论:二价分泌型pcDNA3.1-s2×Aβ42疫苗,可在动物体内产生高效价的特异Aβ抗体.更多还原

【Abstract】 AIM: To construct β-amyloid protein (Aβ _ 42) multivalent recombinant DNA vaccine and to induce the production of Aβ antibody.METHODS: DNA fragment encoding Aβ_ 42 gene w as obtained from Tg2576 mice genome by PCR technique and the signal peptide of I gG к light chain was fused to N-terminal of the Aβ_ 42 gene by primer ext ension.The two fragments of Aβ_ 42 gene were ligased using GSGGSG linkers a nd the product was cloned into pcDNA3.1 vector.The expression vector pcDNA3 .1-Aβ_ 42,secretive univalent vector pcDNA3.1-sAβ_ 42,and secretiv e bivalent vector pcDNA3.1-s2×Aβ_ 42 were constructed respectively.The C OS-7 cells were transfected with these plasmids and the expression of Aβ_ 42 was determined by Western blotting.BALB/c mice were immunized by intramuscula r injection and the titer of antibody was detected by ELISA.The binding of the m ice serum to Aβ plaque of the Tg2576 mouse brain was analyzed by immunohistoche mistry and Aβ_ 42 neutralization test.RESULTS: pcDNA3 .1-Aβ_ 42,pcDNA3.1-sAβ_ 42 and pcDNA3.1-s2×Aβ_ 42 were cor rectly constructed and the expression of Aβ_ 42 peptide in COS-7 cells was confirmed by Western Blotting.After six rounds of booster injection with pcDNA3 .1-Aβ_ 42 in 22 weeks,mice did not generate Aβ antibodies.The injection with pcDNA3.1-sAβ_ 42 generated antibodies less than 1∶100 0 and the injection with pcDNA3.1- s2×Aβ_ 42 produced Aβ antibody a fter the second booster injection,and the titer was higher than 1∶5 000 in 22 wk.Mice serum bound specially to Aβ plaque in the Tg2576 mouse brai n and Aβ_ 42 peptide abrogated the reaction. CONCLUSION: Secretive bivalent vector pcDNA3.1-s2×Aβ_ 42 generates a s ignificant amount of Aβ antibody and demonstrates the functional activity of th ese antibodies.更多还原