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目的观察不同种培养基中重组人色素上皮衍生因子(rPEDF)融合蛋白的表达。方法将前期研究已构建的pET28a-PEDF原核表达重组体转化E.coli BL21大肠杆菌表达宿主菌,酶切鉴定阳性菌落后,分别在M9和LB培养基中用异丙基β-D硫代半乳糖(IPTG,Isopropyl-beta-D-thiogalactoside)诱导表达,SDS-PAGE电泳检测表达的PEDF蛋白,美国I mage-Pro Plus分析系统进行蛋白定量分析。结果 LB和M9培养基中均获得相对分子质量约54×103的rPEDF融合蛋白。但LB培养基获得的是rPEDF融合蛋白的包涵体,目的蛋白占总蛋白含量为21.046%,M9培养基获得的是可溶性的rPEDF的融合蛋白,目的蛋白占总蛋白含量的12.31%。结论不同种培养基中均有rPEDF融合蛋白的表达。
Objective To observe the expression of recombinant human pigment epithelium-derived factor (rPEDF) fusion protein in different kinds of media. Methods The prokaryotic recombinant pET28a-PEDF constructed in the previous study was transformed into E. coli BL21 E.coli and the positive colonies were identified by restriction enzyme digestion. The positive colonies were digested with isopropyl β-D thio-half in M9 and LB medium Induced expression by lactose (IPTG, Isopropyl-beta-D-thiogalactoside), PEDF protein by SDS-PAGE electrophoresis, and protein analysis by I mage-Pro Plus analysis system. Results The rPEDF fusion protein with molecular weight of 54 × 103 was obtained in both LB and M9 medium. However, the inclusion body of rPEDF fusion protein was obtained in LB medium. The total protein content of target protein was 21.046%. The soluble protein of rPEDF was obtained in M9 medium, and the target protein accounted for 12.31% of the total protein content. Conclusion The expression of rPEDF fusion protein was found in different kinds of media.