【摘要】：利用聚合酶鏈?zhǔn)椒磻?yīng)將質(zhì)粒pUC19/rhIFN α-2b上的化學(xué)合成人干擾素α-2b(rhIFN α-2b)基因cDNA擴增并重組至載體pBV220上,構(gòu)建重組質(zhì)粒pBV220/rhIFN α-2b,之后轉(zhuǎn)化大腸桿菌DH5 α。經(jīng)篩選,檢測,測序正確后通過溫度誘導(dǎo)表達重組人干擾素α-2b,通過SDS-PAGE電泳觀察到了特異帶,經(jīng)AlphaImager V5.1軟件分析化學(xué)合成人干擾素α-2b基因表達量最大時表達的人干擾素α-2b可占到菌體總蛋白的36%。Western Blotting檢測可見與IFN α抗體特異性結(jié)合的條帶,并且化學(xué)合成的人干擾素α-2b基因的表達量是天然人干擾素α-2b基因的表達量的約123.5倍。 通過設(shè)計實驗對不同的表達溫度條件及使用不同的培養(yǎng)基對人干擾素α-2b蛋白表達的影響作了比較。 研究不同的表達溫度對人干擾素α-2b蛋白表達的影響時發(fā)現(xiàn)利用LB培養(yǎng)基培養(yǎng),菌體在35℃培養(yǎng)到OD_(600)約0.4時轉(zhuǎn)移到30℃培養(yǎng)50min后立即升溫到42℃誘導(dǎo)表達,表達量最高,可占到菌體總蛋白的約35%。 研究使用不同的培養(yǎng)基培養(yǎng)對人干擾素α-2b蛋白表達的影響時發(fā)現(xiàn)培養(yǎng)及誘導(dǎo)表達溫度相同時,即經(jīng)30℃培養(yǎng),42℃誘導(dǎo)表達,使用培養(yǎng)基成分為1%
[Abstract]:The chemically synthesized human interferon 偽 -2b (rhIFN 偽 -2b) gene on plasmid pUC19/rhIFN 偽 -2b was amplified by polymerase chain reaction and recombined into the vector pBV220. The recombinant plasmid pBV220/rhIFN 偽 -2b was constructed and transformed into E. coli DH5 偽. After screening, detecting and sequencing correctly, the recombinant human interferon 偽 -2b was expressed by temperature induction, and the specific band was observed by SDS-PAGE electrophoresis. The expression of human interferon 偽 -2b at the highest expression level of human interferon 偽 -2b gene was synthesized by AlphaImager V5.1 analytical chemistry. The expression of human interferon 偽 -2b could be detected by 36%.Western Blotting analysis of the total bacterial protein. The specific binding band of human interferon 偽 antibody to IFN 偽 antibody could be found. Moreover, the chemically synthesized human interferon 偽 -2b gene expressed about 123.5 times as much as the natural human interferon 偽 -2b gene. The effects of different expression temperature and medium on the expression of human interferon 偽 -2b protein were compared by designing experiments. When we studied the effect of different expression temperature on the expression of human interferon 偽 -2b protein, we found that the expression of 偽 -2b protein was induced at 35 鈩，

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