摘要
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。
Aim IgMG type of IN-1gene was modify and c DNA seg-ments of IN-1recombinant single chain antibody was developed by gene engineering which is a new way for regeneration promotion of central nerv es and treatment of diffused axonal inj ury.Method s 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.35segments with the length ranging from40-50bp 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 sp licing or-der,the direction and the sequence i n the gene were almost correct except for one nucleotide acid.Conclusion The successful construction of the r e-combinant vector pUC18bearing the c DNA might provide materials for the further research on the IN-1single-chain antibody.
出处
《中国临床康复》
CSCD
2003年第13期1904-1905,共2页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助(39970753)~~
