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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。
Objective To construct small intracellular TAT-6His-mAbA / B, which can specifically antagonize Aβ42 in cells, and provide a new basis for the diagnosis and treatment of AD with intracellular antibody. Methods According to the known sequence of Aβ42 monoclonal antibody provided by GenBank, the variable regions (CDR1 and CDR3) of heavy chain and light chain were selected for conjugation. The primers were designed by using DNASIS computer software and the target fragment mAbA / B was obtained by two PCRs. The target fragment was ligated with pGEM-Teasy vector, restriction endonucleases were used to identify the recombinant plasmids, and Sanger’s single-stranded terminator method was used to detect the nucleotide sequence of the clones. Results The restriction enzyme EcoR I digested pGEM-TEasy / mAbA / B, showing the 132bp or 141bp target fragment, consistent with the theoretical value; DNASIS software analysis and sequencing results and GenBank reported exactly the same results. The recombinant plasmid was identified by restriction endonuclease PamHI and BamH I digestion of pssHG-CMV / TAT-6His-mAbA / B and the fusion gene TAT-6His-mAbA / B fragment of about 400 bp in size was obtained by agarose gel electrophoresis , Consistent with the theoretical value. Conclusion The Aβ42 small intracellular antibody TAT-6His-mAbA / B fragment was successfully cloned by PCR.