CAJ | 학술논문

目的建立能稳定分泌人内皮抑素(hES)的基因工程细胞系,观察内皮抑素(ES)蛋白和hES的表达.方法以hES重组质粒pcDNA3.0(pcDNA3-Endo)为模板,通过聚合酶链反应(PCR)扩增获得hES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体绿色荧光蛋白(pEGFP-N1)质粒中,获得重组质粒pEGFP-N1-Es;利用阳离子脂质体介导将其转染到人胚胎肾细胞(HeK-293)细胞中,G418筛选后得到阳性克隆hES/293,采用蛋白免疫印迹(Western blot)法检测转染细胞上清中ES蛋白的表达;用海澡酸钠壳聚糖(ACA)微囊包裹hES/293细胞,分别收集培养3、7、21、35 d的ACA微囊化hES/293细胞的培养上清液,Western blot法检测包裹后培养液上清中hES的表达.结果重组质粒pEGFP-N1-ES经限制性核对内切酶HindⅢ和限制性核酸内切酶BamH Ⅰ双酶切得到4700碱基对(bp)和600 bp 2条带;PCR扩增出600 bp条带;测序结果与NCBI上序列比对软件(BLAST)比对,同源性达到100%.pEGFP-N1-ES转染HeK-293细胞,经G418筛选后获得阳性克隆,选取筛选的10株单克隆细胞培养上清液进行Western blot分析,在相对分子质量为20×103处出现蛋白条带.在ACA微囊内hES/293细胞随着培养时间的延长,细胞团逐渐长大,充满整个囊内空间.培养3、7、21、35 d时,在相对分子质量为20×103处出现蛋白条带.结论重组pEGFP-N1-ES真核表达载体构建正确,转染HeK-293细胞后可有效的表达hES蛋白,并能分泌到细胞外;微囊化hES/293细胞产生的ES蛋白可以自由扩散出微囊膜外,并呈持续性表达.
목적 건립능은정분비인내피억소(hES)적기인공정세포계,관찰내피억소(ES)단백화hES적표체.방법 이hES중조질립pcDNA3.0(pcDNA3-Endo)위모판,통과취합매련반응(PCR)확증획득hES기인편단,차재기인전가신호태서렬,장기정향삽입진핵표체재체록색형광단백(pEGFP-N1)질립중,획득중조질립pEGFP-N1-Es;이용양리자지질체개도장기전염도인배태신세포(HeK-293)세포중,G418사선후득도양성극륭hES/293,채용단백면역인적(Western blot)법검측전염세포상청중ES단백적표체;용해조산납각취당(ACA)미낭포과hES/293세포,분별수집배양3、7、21、35 d적ACA미낭화hES/293세포적배양상청액,Western blot법검측포과후배양액상청중hES적표체.결과 중조질립pEGFP-N1-ES경한제성핵대내절매HindⅢ화한제성핵산내절매BamH Ⅰ쌍매절득도4700감기대(bp)화600 bp 2조대;PCR확증출600 bp조대;측서결과여NCBI상서렬비대연건(BLAST)비대,동원성체도100%.pEGFP-N1-ES전염HeK-293세포,경G418사선후획득양성극륭,선취사선적10주단극륭세포배양상청액진행Western blot분석,재상대분자질량위20×103처출현단백조대.재ACA미낭내hES/293세포수착배양시간적연장,세포단축점장대,충만정개낭내공간.배양3、7、21、35 d시,재상대분자질량위20×103처출현단백조대.결론 중조pEGFP-N1-ES진핵표체재체구건정학,전염HeK-293세포후가유효적표체hES단백,병능분비도세포외;미낭화hES/293세포산생적ES단백가이자유확산출미낭막외,병정지속성표체.
Objective To microencapsulate a genetically engineered cell line which stably secrete human endostatin (hES). Methods Endostatin gene fragment was amplified from plasmid pcDNA3-Endo by polymerase chain reaction, and inserted into mammalian eukaryotic expression vector pEGFP-N1, resulting into recombinant plasmid pEGFP-N1-ES. Hek-293 cells were transfected with pEGFP-N1-ES via cationic liposome and selected by G418, and were measured by Western blot for endostatin protein expression. The hES/293 cells were further entrapped by alginate-chitosan-alginate (ACA) microcapsules, and the expression of endostatin in the supernatant of cultured hES/293 cell microcapsules was examined by western blot at different time points. Results Recombinant plasmid pEGFP-N1-endostatin was digested by HindⅢ and BamHI, and resulted into 2 DNA fragments of 7 kb and 600 bp. The sequence of the 600 bp fragment was identical to human endostatin. Western blot of the supernatant of cultured hES/293 cells or hES/293 cell microcapsules detected a positive band with the relative molecular mass of 20 × 103. Conclusion The hES protein was expressed in HeK-293 transfected with pEGFP-N1-endostatin, and secreted to the culture medium, and can freely diffused outside the micro-capsule.