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目的:研究不同浓度的PEG8000对融合蛋白ELP30-Trx相变温度的影响。方法:将硫氧还蛋白(Thioredoxin,Trx)基因克隆到pET28-ELP30-Trx表达载体中,经IPTG诱导在宿主菌E.coli BLR(DE3)中表达ELP30-Trx,通过可逆相变循环(Inverse transition cycling,ITC)纯化融合蛋白。测量相变温度(T t),并检测不同浓度的PEG8000对ELP30-Trx T t的影响。结果:经酶切及测序鉴定证实成功构建了ELP30-Trx表达载体,经Westen-Blot检测表明成功表达ELP30-Trx融合蛋白。经ITC纯化后的ELP30-Trx纯度可达95%以上。5%、10%、15%、20%的PEG8000(W/V)使终浓度为25μmol/L ELP30-Trx的T t从37.9℃分别降低至34.6℃、28.5℃、14.0℃、6.0℃。结论:成功构建、表达融合蛋白ELP30-Trx,PEG8000能显著降低ELP30-Trx相变温度。通过对类弹性蛋白的研究,为类弹性蛋白的扩大应用提供理论依据。
Objective: To study the effect of different concentrations of PEG8000 on the phase transition temperature of ELP30-Trx fusion protein. Methods: The Thioredoxin (Trx) gene was cloned into pET28-ELP30-Trx expression vector and induced by IPTG to express ELP30-Trx in E. coli BLR (DE3) transition cycling, ITC). The phase transition temperature (T t) was measured and the effect of different concentrations of PEG 8000 on ELP30-Trx T t was measured. Results: The ELP30-Trx expression vector was successfully constructed by restriction endonuclease analysis and sequencing. The expression of ELP30-Trx fusion protein was confirmed by Westen-Blot assay. ELP30-Trx purified by ITC can reach more than 95% purity. The T t of ELP30-Trx at a final concentration of 25 μmol / L was reduced from 37.9 ° C to 34.6 ° C, 28.5 ° C, 14.0 ° C, 6.0 ° C at 5%, 10%, 15%, 20% PEG8000 (WV). Conclusion: The successful construction and expression of the fusion proteins ELP30-Trx and PEG8000 can significantly reduce the phase transition temperature of ELP30-Trx. Through the study of elastin, provide a theoretical basis for the expansion of the application of elastin.