CAJ | 학술논문

目的构建表达IL - 1ra的慢病毒载体,为神经前体细胞(NPCs)的基因修饰移植治疗脊髓损伤的研究提供基础.方法从IL - 1ra质粒中克隆出IL -1ra基因,下游融合IRES2 - EGFP,装载于pLenti6.3 V5 - DEST中,获得含目的序列IL1ra - IRES2 - EGFP的慢病毒质粒,测序并包装；病毒感染HEK293细胞,通过GFP表达水平测定病毒滴度.并转染NPCs,观察NPCs的生长情况.结果测序证实,用引物CMV -F和IRES2 -R扩增的质粒中IL -1ra的DNA序列与Genebank中序列完全一致；慢病毒悬液的滴度为3.5×106 TU/ml；NPCs转染重组慢病毒后且未出现生长特性的改变.结论成功构建了表达IL - 1ra的慢病毒载体pLenti6.3- IL1ra - IRES2 - EGFP.
목적 구건표체IL - 1ra적만병독재체,위신경전체세포(NPCs)적기인수식이식치료척수손상적연구제공기출.방법 종IL - 1ra질립중극륭출IL -1ra기인,하유융합IRES2 - EGFP,장재우pLenti6.3 V5 - DEST중,획득함목적서렬IL1ra - IRES2 - EGFP적만병독질립,측서병포장；병독감염HEK293세포,통과GFP표체수평측정병독적도.병전염NPCs,관찰NPCs적생장정황.결과 측서증실,용인물CMV -F화IRES2 -R확증적질립중IL -1ra적DNA서렬여Genebank중서렬완전일치；만병독현액적적도위3.5×106 TU/ml；NPCs전염중조만병독후차미출현생장특성적개변.결론 성공구건료표체IL - 1ra적만병독재체pLenti6.3- IL1ra - IRES2 - EGFP.
Objective To construct the lentivirus vector expressing IL - 1 ra,which will be used in the study of gene modification and transplantation of neural precursor cells (NPCs).Methods IL -1ra cDNA was cloned from IL - 1ra plasmid by PCR,fused with IRES2 - EGFP downstream,and loaded to pLenti6.3 V5 - DEST.After sequencing and package were completed,the lentivirus plasmid was named as pLenti6.3-IL1ra -IRES2 - EGFP and was used to infect HEK293 cells.The titer of virus was tested according to the expressing level of EGFP.Results DNA sequencing analysis confirmed that the DNA sequence of IL -lra in plasmid amplified from primers CMV - F and IRES2 - R was the same as which was adopted in Genebank.The titer of the virus suspension was 3.5 × 106 TU/ml.There was not obvious change of growth characteristics in NPCs after which were transinfected with recombinant lentivirus.Conclusion Lentivirus vector expressing IL -1ra was constructed successfully.