【摘要】 目的抗NI-35抗体(anti-NI-35antibody)作为神经再生抑制因子(NI-35)的拮抗剂对神经再生促进作用的研究是近年的热点,研究表明,抗NI-35抗体能促进体内外受损伤神经元的存活和突起生长。我们重新设计并人工合成抗NI-35重组单链抗体(anti-NI-35-scfv)的cDNA克隆构建其表达载体,在原核系统中实现初步表达。方法参照genebank中发表的抗NI-35抗体的轻链重链序列,重新设计适于在大肠杆菌中表达的目的基因片段,将该基因双链分成35个小片段合成,经退火、复性连接成目的片段后,克隆到经过BamHI和HindIII双酶切的克隆载体pUC18中,并转化大肠杆菌DH5a,抽提重组子pUC18/744进行克隆PCR、酶切鉴定及测序分析。结果测序结果证明获得的基因序列与实验设计仅差一个碱基。结论正确合成了抗NI-35重组单链抗体(anti-NI-35-single-chainantibody)为抗NI-35抗体应用于治疗弥漫性轴索损伤提供了实验基础。更多还原

【Abstract】 Objective CNS myelin and oligodendrocyte membranes contain two minor proteins with strong inhibitory effects on growing neuritisneurite growth inhibiters NI-35 and NI-250.Monoclonal antibody anti-NI-35 was obtained that neutralize this activity in CNS. We construct and express a recombinant cloning vector bearing the chemically synthesized cDNA of the recombinant anti-NI-35 single-chain antibody. Method 35 segments with the length ranging from 4050 bp were assembled in only one step by a PCR approach. The entire gene was cloned into pUC18 plasmid .Determined by auto-sequencing the target gene was cloned into prokaryotic expression vector pET28a(+)in fusion form and transformed into E.coli BL21. Result Sequence analysis showed that the splicing order the direction and the sequence in the gene were almost correct.SDS-PAGE analysis showed that a new protein band with molecule weight of 31kD appeared as the expected size. Conclusion The successful construction and expression of the recombinant vector pET-28a(+) bearing the cDNA might provide material for the further research about the anti-NI-35 single-chain antibody .更多还原