蜂毒肽/变构hIL-2融合蛋白的原核表达及其对宿主菌生长的影响

目的构建蜂毒肽(Melittin)与变构hIL-2融合基因原核表达质粒,并检测表达的融合蛋白对宿主菌E.coli生长的影响。方法以质粒pGEX-4T-2/Melittin-IL-2(88Arg)为模板,通过PCR定点诱变为Melittin-IL-2(88Arg,125Ala),将PCR产物和pET-15b载体分别经双酶切后连接,构建重组表达质粒pET-15b/M-IL-2(88Arg,125Ala),转化E.coliBL21(DE3),IPTG诱导表达。SDS-PAGE及ELISA分析表达产物;检测诱导不同时间重组菌的A600值,绘制生长曲线,并进行活菌计数。结果PCR扩增的目的片段长542bp,重组表达质粒测序分析证明目的基因如预期突变;ELISA可检测到目的蛋白表达,但表达量较低;SDS-PAGE分析未见目的条带;诱导表达4h,重组菌A600值由0.8下降至0.6;诱导2h,活菌计数由108个/ml降至104个/ml。结论已成功构建了原核表达质粒pET-15b/M-IL-2(88Arg,125Ala),表达的融合蛋白可能对宿主菌具有毒性,从而杀伤宿主菌。     

Structural and functional behavior of biologically active monomeric melittin

Melittin is a well-known water-soluble toxic peptide present in bee venom of Apis mellifera, capable of interacting with and disrupting cell membranes thus producing many effects on living cells. Additionally, melittin induces activation of phospholipases and calmodulin upon interaction with cellular membranes. The conformation and aggregation state adopted by melittin in solution depends on several factors including the peptide concentration, ionic strength, pH and the nature of the ions in the aqueous medium. Such conformational dependence on the peptide environment gives new insights over the currently available 3D structures of melittin and, ultimately, over its biologically functional unit. Based on crystallographic data, the melittin tetramer has been proposed as its bioactive form. Contrarily to such data, we show in this work the results obtained from molecular dynamics simulations, which clearly indicate that the tetrameric organization of melittin is not stable under biological conditions dissociating after 2.5 ns through a 10 ns trajectory. We found that the tetrameric form of melittin is stable only in conditions of high pH and high peptide concentration in the molecular dynamics simulations. Moreover, when in plasma melittin appears to be a random coil monomer, folding only upon interaction with biological membranes. In summary, these findings elucidate several properties of melittin structure and dynamics, projecting significant implications in the study of its biological function.     

Promoting effect of ethanol on dewetting transition in the confined region of melittin tetramer

To study the influence of ethanol molecules on the melittin tetramer folding,we investigated the dewetting transition of the melittin tetramer immersed in pure water and 8%aqueous ethanol solution(mass fraction) by the molecular dynamics simulations.We found that the marked dewetting transitions occurred inside a nanoscale channel of the melittin tetramer both in pure water and in aqueous ethanol solution.Also,ethanol molecules promoted this dewetting transition.We attributed this promoting effect to ethanol molecules which prefer to locate at the liquidvapor interface and decrease the liquid-vapor surface energy.The results provide insight into the effect of ethanol on the water dewetting phenomena.     

蜂毒肽高效液相色谱测定

本文应用高效液相色谱对蜂毒中多肽溶血毒进行了定量测定。标准偏差<1.28％。平均回收率>96％。