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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。
OBJECTIVE: To improve the gene quality of IgMG-type IN-1 by genetic engineering to obtain an IN-1 recombinant single-chain antibody (scFv) cDNA gene fragment to promote the regeneration of impaired central nervous system and to treat diffuse axonal injury A new way. Methods The host bacteria was E. coli DH5a and the cloned plasmid was pUC18. With reference to the light chain heavy chain sequence of IN-1 antibody published in genebank, the target gene fragment suitable for expression in E. coli was redesigned, and the gene was double-stranded and divided into 35 small fragments for synthesis. After annealing and refolding, The fragment was cloned into the cloned vector pUC18 digested with BamHI and HindIII and transformed into E. coli DH5a. The recombinant plasmid pUC18 / 744 was extracted for cloning PCR, restriction enzyme digestion and sequencing analysis. Results Sequencing results showed that the obtained gene sequence was only one base away from the experimental design. Conclusions The cDNA of IN-1 recombinant single chain antibody (IN-1-scFv) was designed and synthesized correctly, which laid the foundation for further study of its biological activity. It also opened up a new idea for antibody engineering to treat diffuse axonal injury.