CAJ | 학술논문

目的利用基因工程技术,对 IgMG型的 IN-1的基因进行改良,以得到 IN-1重组单链抗体( scFv) cDNA基因片段为促使受损的中枢神经再生和治疗弥漫性轴索损伤开辟一个崭新途径.方法宿主菌为大肠杆菌 DH5a,克隆质粒为 pUC18.参照 genebank中发表的 IN-1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.结论正确设计并合成了 IN-1重组单链抗体( IN-1-scFv)的 cDNA,为深入研究其生物活性奠定了基础,也为应用抗体工程治疗弥漫性轴索损伤开创了一个新思路.
목적이용기인공정기술,대 IgMG형적 IN-1적기인진행개량,이득도 IN-1중조단련항체( scFv) cDNA기인편단위촉사수손적중추신경재생화치료미만성축색손상개벽일개참신도경.방법숙주균위대장간균 DH5a,극륭질립위 pUC18.삼조 genebank중발표적 IN-1항체적경련중련서렬,중신설계괄우재대장간균중표체적목적기인편단,장해기인쌍련분성 35개소편단합성,경퇴화、복성련접성목적편단후,극륭도경과 BamHI화 HindIII쌍매절적극륭재체 pUC18중,병전화대장간균 DH5a,추제중조자 pUC18/744진행극륭 PCR、매절감정급측서분석.결과측서결과증명획득적기인서렬여실험설계부차일개감기.결론정학설계병합성료 IN-1중조단련항체( IN-1-scFv)적 cDNA,위심입연구기생물활성전정료기출,야위응용항체공정치료미만성축색손상개창료일개신사로.
Aim IgMG type of IN-1 gene was modify and cDNA segments of IN-1 recombinant single chain antibody was developed by gene engineering which is a new way for regeneration promotion of central nerves and treatment of diffused axonal injury.Methods Host bacterium was E. Coli DH5a,and cloning plasmid was pUC18.Referring to light and heavy chain published in GeneBank,we redesigned gene segments which were suitable to express in E. Coli.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 cloning plasmid vector pUC18.The sequence of the cloned cDNA was confirmed by DNA sequncing, clone PCR and restriction enzymes analysis.Results Sequence analysis showed that the splicing order, the direction and the sequence in the gene were almost correct except for one nucleotide acid.Conclusion The successful construction of the recombinant vector pUC18 bearing the cDNA might provide materials for the further research on the IN-1 single-chain antibody.