Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells...Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA （IFN αA） gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10<sup>10</sup> units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.展开更多
应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动...应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。展开更多
目的克隆信号转导与转录活化子1(signal transducer and activators of transcription,STAT)基因片段并构建其真核重组表达质粒。通过研究Stat1 mRNA序列各组成部分与Stat1基因翻译调控的关系,探讨了Stat1蛋白翻译调控机制。方法采用DN...目的克隆信号转导与转录活化子1(signal transducer and activators of transcription,STAT)基因片段并构建其真核重组表达质粒。通过研究Stat1 mRNA序列各组成部分与Stat1基因翻译调控的关系,探讨了Stat1蛋白翻译调控机制。方法采用DNA重组技术,将用SMART RACE法筛选出的Stat1基因片段克隆到pT-Adv载体,然后将其亚克隆到pFLAG-CMV-2真核表达载体上,构建Stat1 cDNA重组质粒。分别采用磷酸钙转染法、DEAE-葡聚糖转染法和脂质体介导转染法将其转染Hela细胞,并对3种方法的转染效率及各重组Stat1质粒mRNA和蛋白质表达的差异进行比较,以研究Stat1 mRNA结构对Stat1表达的影响。结果构建了CS,C3S,CS/3S,5SFC3S,5SFCS和CL/3L共6个重组Stat1质粒。通过比较发现,磷酸钙转染法、DEAE-葡聚糖转染法的转染效率明显低于脂质体介导转染法,转染的质粒均得到清晰的mRNA和蛋白质表达信号。用INF-α刺激转染5SFC3S重组Stat1质粒的Hela细胞,发现刺激组与空白组Stat1 mRNA转录水平和蛋白质表达量无明显差异,蛋白质合成效率无降低,此结果与野生型Stat1对INF-α刺激不同。结论将重组Stat1质粒转染Hela细胞后,在短暂表达条件下,重组Stat1质粒与野生型Stat1对INF-α刺激反应不同,其原因尚有待进一步研究。展开更多
文摘Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA （IFN αA） gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10<sup>10</sup> units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.
文摘应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。
