按大肠杆菌偏爱密码子化学合成人干扰素α-2b 基因在大肠杆菌中高效表达的研究
发布时间：2018-10-24 11:46
【摘要】：利用聚合酶链式反应将质粒pUC19/rhIFN α-2b上的化学合成人干扰素α-2b(rhIFN α-2b)基因cDNA扩增并重组至载体pBV220上,构建重组质粒pBV220/rhIFN α-2b,之后转化大肠杆菌DH5 α。经筛选,检测,测序正确后通过温度诱导表达重组人干扰素α-2b,通过SDS-PAGE电泳观察到了特异带,经AlphaImager V5.1软件分析化学合成人干扰素α-2b基因表达量最大时表达的人干扰素α-2b可占到菌体总蛋白的36%。Western Blotting检测可见与IFN α抗体特异性结合的条带,并且化学合成的人干扰素α-2b基因的表达量是天然人干扰素α-2b基因的表达量的约123.5倍。 通过设计实验对不同的表达温度条件及使用不同的培养基对人干扰素α-2b蛋白表达的影响作了比较。 研究不同的表达温度对人干扰素α-2b蛋白表达的影响时发现利用LB培养基培养,菌体在35℃培养到OD_(600)约0.4时转移到30℃培养50min后立即升温到42℃诱导表达,表达量最高,可占到菌体总蛋白的约35%。 研究使用不同的培养基培养对人干扰素α-2b蛋白表达的影响时发现培养及诱导表达温度相同时,即经30℃培养,42℃诱导表达,使用培养基成分为1%
[Abstract]:The chemically synthesized human interferon 伪 -2b (rhIFN 伪 -2b) gene on plasmid pUC19/rhIFN 伪 -2b was amplified by polymerase chain reaction and recombined into the vector pBV220. The recombinant plasmid pBV220/rhIFN 伪 -2b was constructed and transformed into E. coli DH5 伪. After screening, detecting and sequencing correctly, the recombinant human interferon 伪 -2b was expressed by temperature induction, and the specific band was observed by SDS-PAGE electrophoresis. The expression of human interferon 伪 -2b at the highest expression level of human interferon 伪 -2b gene was synthesized by AlphaImager V5.1 analytical chemistry. The expression of human interferon 伪 -2b could be detected by 36%.Western Blotting analysis of the total bacterial protein. The specific binding band of human interferon 伪 antibody to IFN 伪 antibody could be found. Moreover, the chemically synthesized human interferon 伪 -2b gene expressed about 123.5 times as much as the natural human interferon 伪 -2b gene. The effects of different expression temperature and medium on the expression of human interferon 伪 -2b protein were compared by designing experiments. When we studied the effect of different expression temperature on the expression of human interferon 伪 -2b protein, we found that the expression of 伪 -2b protein was induced at 35 鈩，
本文编号：2291312
[Abstract]:The chemically synthesized human interferon 伪 -2b (rhIFN 伪 -2b) gene on plasmid pUC19/rhIFN 伪 -2b was amplified by polymerase chain reaction and recombined into the vector pBV220. The recombinant plasmid pBV220/rhIFN 伪 -2b was constructed and transformed into E. coli DH5 伪. After screening, detecting and sequencing correctly, the recombinant human interferon 伪 -2b was expressed by temperature induction, and the specific band was observed by SDS-PAGE electrophoresis. The expression of human interferon 伪 -2b at the highest expression level of human interferon 伪 -2b gene was synthesized by AlphaImager V5.1 analytical chemistry. The expression of human interferon 伪 -2b could be detected by 36%.Western Blotting analysis of the total bacterial protein. The specific binding band of human interferon 伪 antibody to IFN 伪 antibody could be found. Moreover, the chemically synthesized human interferon 伪 -2b gene expressed about 123.5 times as much as the natural human interferon 伪 -2b gene. The effects of different expression temperature and medium on the expression of human interferon 伪 -2b protein were compared by designing experiments. When we studied the effect of different expression temperature on the expression of human interferon 伪 -2b protein, we found that the expression of 伪 -2b protein was induced at 35 鈩，
本文编号：2291312
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