CAJ | 학술논문

背景：抑制椎间盘细胞的凋亡可以延缓椎间盘的退变，而生存素具有调节细胞增殖和抗凋亡功能。 　　目的：构建人生存素基因的慢病毒载体。方法：应用全基因合成技术合成人生存素基因(BIRC5)，通过 PCR扩增目的基因并对 PCR 结果进行电泳分析。将目的基因克隆到慢病毒表达质粒构建重组慢病毒质粒 Lenti-BIRC5。将重组的慢病毒质粒转化细菌感受态细胞，PCR鉴定阳性的克隆进行基因测序。将含有目的基因的慢病毒质粒转染293T细胞，应用Western blot技术对重组慢病毒载体 Flag-Survivin 融合蛋白的表达进行检测。 　　结果与结论：PCR鉴定及基因测序结果显示成功构建了含有人Survivin基因的慢病毒表达载体，Western blot检测结果显示目的基因在体外培养细胞中转染成功并且过表达。说明慢病毒表达载体Lenti-BIRC5构建成功，这为下一步研究Survivin在人髓核细胞中的抗凋亡作用提供了载体。
배경：억제추간반세포적조망가이연완추간반적퇴변，이생존소구유조절세포증식화항조망공능。 　　목적：구건인생존소기인적만병독재체。방법：응용전기인합성기술합성인생존소기인(BIRC5)，통과 PCR확증목적기인병대 PCR 결과진행전영분석。장목적기인극륭도만병독표체질립구건중조만병독질립 Lenti-BIRC5。장중조적만병독질립전화세균감수태세포，PCR감정양성적극륭진행기인측서。장함유목적기인적만병독질립전염293T세포，응용Western blot기술대중조만병독재체 Flag-Survivin 융합단백적표체진행검측。 　　결과여결론：PCR감정급기인측서결과현시성공구건료함유인Survivin기인적만병독표체재체，Western blot검측결과현시목적기인재체외배양세포중전염성공병차과표체。설명만병독표체재체Lenti-BIRC5구건성공，저위하일보연구Survivin재인수핵세포중적항조망작용제공료재체。
BACKGROUND:Inhibiting the apoptosis of intervertebral disc cel s can postpone the degenerative process of intervertebral disc. Survivin has a strong function of regulating cel proliferation and anti-apoptosis. OBJECTIVE:To construct and identify the lentiviral vector encoding survivin gene of human. METHODS:The survivin gene of human (BIRC5) was synthesized through the gene synthesis technology, amplified by PCR and analyzed by electrophoresis. The target gene was cloned into lentiviral expression plasmid to obtain the recombinant lentiviral vector Lenti-BIRC5. After transformation into competent E. coli cel s, the candidate clones were identified by PCR firstly. The positive clones were identified by gene sequencing. The lentivirus plasmid containing target gene was transfected into 293T cel s, and the expression of recombinant lentiviral vector Flag-Survivin fusion protein was detected through western blot analysis. RESULTS AND CONCLUSION:The PCR results of electrophoresis and DNA sequencing showed that lentiviral vector containing human survivin gene was constructed successful y. Western blot analysis results showed that the target gene was transfected successful y and over-expressed in cultured cel s. The lentiviral expression vector of human survivin gene Lenti-BIRC5 was constructed successful y, which lays a foundation for the study addressing the anti-apoptotic effects of survivin on human nucleus pulposus cel s.