CAJ | 학술논문

目的联合应用 LR 克隆酶和链霉菌噬菌体ΦC31整合酶系统，建立一种获得位点特异整合型微环 DNA 的方法，为该载体在转基因研究中的应用提供科学资料。 　　方法应用分子克隆技术，在目的基因表达盒及链霉菌attB 位点两端接入λattR 和λattL 片段，随后插入ΦC31整合酶基因表达盒，构建可获得微环 DNA 的亲本质粒。该亲本质粒上的λattL 和λattR 序列可在 LR 克隆酶的催化下重组生成表达ΦC31整合酶的微质粒和含有目的基因表达盒及 attB 位点的微环 DNA。以限制性内切酶酶切、定性以及定量 PCR 分析其重组效率。并将未经纯化的 LR重组体系转染 HeLa 细胞，以流式细胞技术、克隆计数、ELISA 等方法检测其转染率、整合率及目的蛋白表达水平，并与传统质粒进行比较。 　　结果 LR 克隆酶可有效催化亲本质粒的重组，重组率达80%以上。重组体系中的微环 DNA 和微质粒无需纯化即可成功转染 HeLa 细胞，且其转染率、整合率以及目的蛋白表达水平均高于传统质粒。 　　结论建立了一种高效、快速获得位点特异整合型微环DNA 的方法。
목적연합응용 LR 극륭매화련매균서균체ΦC31정합매계통，건립일충획득위점특이정합형미배 DNA 적방법，위해재체재전기인연구중적응용제공과학자료。 　　방법응용분자극륭기술，재목적기인표체합급련매균attB 위점량단접입λattR 화λattL 편단，수후삽입ΦC31정합매기인표체합，구건가획득미배 DNA 적친본질립。해친본질립상적λattL 화λattR 서렬가재 LR 극륭매적최화하중조생성표체ΦC31정합매적미질립화함유목적기인표체합급 attB 위점적미배 DNA。이한제성내절매매절、정성이급정량 PCR 분석기중조효솔。병장미경순화적 LR중조체계전염 HeLa 세포，이류식세포기술、극륭계수、ELISA 등방법검측기전염솔、정합솔급목적단백표체수평，병여전통질립진행비교。 　　결과 LR 극륭매가유효최화친본질립적중조，중조솔체80%이상。중조체계중적미배 DNA 화미질립무수순화즉가성공전염 HeLa 세포，차기전염솔、정합솔이급목적단백표체수평균고우전통질립。 　　결론건립료일충고효、쾌속획득위점특이정합형미배DNA 적방법。
Objective To develop an improved method for generation of site-specific integration minicircle DNA vector by combination of LR clonase from E. coliλphage with streptomyces phageΦC31 integrase system, and to test the transfection efficiency, integration rate, and protein expression level of the minicircle DNA vector by transfecting HeLa cells. Methods A parental plasmid containing ΦC31 integrase gene expression cassette, attB, λattR and λattL was constructed. The recombination ofλattR andλattL in the parental plasmid was mediated by LR clonase, which generated a minicircle DNA containing gene of interest and a miniplasmid containing ΦC31 integrase gene expression cassette. The LR recombination products were transfected to HeLa cells without purification. Transfection efficiency and integration rate of the minicircle DNA and control plasmid were detected 24 hours and 14 days after transfection, respectively. GFP expression level of GFP-positive colonies picked under fluorescence microscopy was evaluated by FACS analysis. After the gene of interest in minicircle DNA was replaced from EGFP to hTPO, hTPO expression level in various time points after transfection was measured by ELISA, and compared with the control plasmid. Results Restriction enzyme digestion, qualitative and quantitative PCR results indicated that LR clonase mediated the recombination ofλattR andλattL in the parental plasmid with high efficiency of more than 80%. The LR recombination products could be successfully transfected into HeLa cells without purification. Flow cytometry, colonies counting and ELISA results confirmed that the minicircle DNA vectors were with higher transfection efficiency, integration rate, and protein expression level than control plasmids. Conclusion An improved method to generate site-specific integration minicircle DNA was successfully established. By comparing with control plasmids which contain bacterial backbone, minicircle DNA vectors can be transfected into HeLa cells with higher transfection efficiency and higher protein expression level.