| 摘要: |
| 【目的】将已经成功构建的AcMNPV-lef-10表达载体在原核和真核表达系统中表达，研究其形成高分子复合物的特性，为揭示其高分子复合物的形成机理奠定基础。【方法】将先前成功构建的pTriEx-lef-10、pTriEx-lef-10-1-52、pTriEx-lef-10-27-78重组质粒分别转化至大肠杆菌BL21 (DE3)和Sf 9细胞中诱导表达，产物经纯化、浓缩后，进行SDS-PAGE和Western blot检测分析；利用SDD-AGE技术估测LEF-10蛋白的分子质量；用透射电子显微镜分别观察原核和真核系统表达的LEF-10蛋白的外部形态。【结果】Western blot分析显示，LEF-10蛋白能形成高分子复合物，而C末端、N末端缺失和C末端融合GFP都可以阻断或部分阻断LEF-10高分子复合物的形成；苯酚 氯仿 异戊醇能部分打开LEF-10蛋白高分子复合体；利用SDD-AGE技术初步估测LEF-10高分子复合体分子质量集中在520～3 250 ku；透射电子显微镜观察原核和真核系统表达的LEF-10，可见蛋白复合体大小不均一，呈现球形或者近球形。【结论】LEF-10蛋白在原核和真核表达系统中，都会形成高分子复合体，该复合体在强变性剂作用下，也不能完全打开，推测该复合体可能以共价键形式结合。 |
| 关键词: AcMNPV LEF-10 原核表达 真核表达 |
| DOI： |
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| 基金项目:陕西省自然科学基础研究计划项目（2011JM3002） |
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| Characteristics of insoluble complex formed by AcMNPV LEF-10 |
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ZHU Kong-li,ZHAO Yu,JIANG Xiang,et al
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| Abstract: |
| 【Objective】AcMNPV lef-10 expression vector was expressed in prokaryotic and eukaryotic expression systems and the characteristics of the formed polymer compounds were investigated to reveal their formation mechanism.【Method】Previously constructed pTriEx-lef-10,pTriEx-lef-10-1-52,and pTriEx-lef-10-27-78 recombinant plasmids were transformed into BL21 (DE3) cells and Sf 9 cells for protein expression,respectively.After being purified and concentrated,LEF-10 proteins were analyzed by SDS-PAGE and Western blot.The molecular weights of LEF-10 proteins were estimated using SDD-AGE and LEF-10 proteins expressed by prokaryotic and eukaryotic expression systems were observed using transmission electron microscopy (TEM).【Result】Western blot showed that LEF-10 proteins could form high polymer compounds,and C-terminal and N-terminal truncation and C-terminal fusing of GFP could prevent or partly prevent LEF-10 from forming high polymer compounds.The high molecular complexes could be partly disassembled by phenol-chloroform-isoamyl alcohol.Molecular weights of LEF-10 proteins were estimated to be 520-3 250 ku by SDD-AGE.By transmission electron microscopy (TEM),LEF 10 protein complexes were observed as sphere or near sphere with various sizes.【Conclusion】LEF-10 proteins can form high polymer compounds in both prokaryotic and eukaryotic expression systems.As the high polymer compounds could not be completely opened by strong denaturants,it was speculated that covalent bonds could be involved in the formation of complexes. |
| Key words: AcMNPV LEF-10 prokaryotic expression eukaryotic expression |
