【摘要】 目的构建能够在细胞内特异性拮抗Aβ42的小分子细胞内抗体TAT-6His-mAbA/B,为细胞内抗体技术用于AD的诊断和治疗提供新依据。方法根据GenBank提供的Aβ42单克隆抗体的已知序列,选取重链和轻链可变区(CDR1和CDR3)进行偶联,应用DNASIS计算机软件设计引物,通过两次PCR获得目的片段mAbA/B。将目的片段与pGEM-Teasy载体相连,限制性内切酶酶切鉴定重组质粒,Sanger单链末端终止法测出克隆的核苷酸序列。结果限制性内切酶EcoR I酶切pGEM-TEasy/mAbA/B,可见132bp或141bp的目的片段,与理论值一致;DNASIS软件分析测序结果与GenBank所报道的结果完全一致。限制性内切酶PamHI和BamH I联合酶切pssHG-CMV/TAT-6His-mAbA/B,琼脂糖凝胶电泳鉴定重组质粒,得到大小约为400bp的融合基因TAT-6His-mAbA/B目的片段,与理论值一致。结论通过PCR方法成功克隆了Aβ42小分子细胞内抗体TAT-6His-mAbA/B片段。更多还原

【Abstract】 Objective To construct the intracellular antibodies of Aβ42 TAT-6His-mAbA/B and lay a new foundation for the diagnosis and treatment of AD.Methods Based on GenBank providing the sequence monoclone antibody of Aβ42,we utilizes variable CDRs(CDR1 and CDR3)by coupling them with special linker.Analyzing and designing the primers with DNASIS software through double PCR for the target genes mAbA/B.Ligating the target genes with vector pGEM-Teasy and identifing the recombined plasmid with restriction enzymes,they were tested by Sanger single termination the sequence of the target DNA.Results After digestion of pGEM-T Easy/mAbA/B by restriction enzyme EcoR I,the purpose fragments of 132bp or 141bp were got,consistenting with the theoretical value.The results of DNASIS sequencing analysis software were consistent with that reported in GenBank.Digesting the plasmid pssHG-CMV/TAT-6His-mAbA/B with restriction enzyme BamHI and PamHI and agarose gel electrophoresis of recombinant plasmid,we obtained the gene TAT-6His-mAbA/B fragment of about 400bp,consistenting with the theoretical value.Conclusion The miniantibodies of Aβ42(TAT-6His-mAbA/B)can be constructed by PCR successfully.更多还原