论文摘要
由活化的酚氧化酶原所诱导激活的黑化反应能够结合并杀死入侵的外源微生物,是节肢动物先天性免疫反应重要的组成部分。但是如若体内生成过量的醌或者全身黑化反应过度就会对宿主产生毒害,因此黑化反应应有严格的调控体系。但是对于在体内如何调节控制酚氧化酶所诱导黑化反应的分子机制却了解甚少。在这篇论文中通过生化实验证明,在黄粉虫体内Toll途径中能够切割加工酶原Sp(?)tzle的关键酶—Sp(?)tzle加工酶(Tm-SPE)能把分子量为79 kDa的黄粉虫酚氧化酶原(Tm-proPO)切割成分子量分别为76 kDa,46 kDa和27 kDa的片断,可是这些片断都没有产生黑色素的能力。但是如果把Tm-proPO,活性形式的Tm-SPE及黄粉虫发夹样结构域丝氨酸蛋白酶同系物1(TmSPH1)的酶原形式混和在一起进行孵育,则能产生由分子量为76 kDa的Tm-PO和Tm-SPH1的活性形式所组成的高分子量的黑化复合物。而且如果把Tm-SPH1换成Tm-SPH2就不能产生黑化。这种复合物能够在革兰阴性和革兰阳性的细菌表面合成大量的黑化产物因而具有明显的杀菌能力。综上所诉,本研究结果表明在黄粉虫体内Toll途径与酚氧化酶原激活系统这两种主要的先天性免疫反应路径可能是由同一种丝氨酸蛋白酶级联系统所激活的。且在酚氧化酶所诱导的黑化反应中Tm-SPH1作为必需的调节因子能够严格的调控酚氧化原酶联反应的激活以免由于全身过度黑化给宿主带来致命的伤害。
论文目录
LIST OF FIGURESABBREVIATIONSABSTRACT(IN ENGLISH)ABSTRACT(IN CHINESE)1.INTRODUCTION2.MATERIALS AND METHODS2.1 Materials2.1.1 Insects and collection of hemolymph2.1.2 Reagents and buffers2.2 Measurement of amidase activity2.3 Determination of melanin synthesis2.4 Amino acid sequencing2.5 Antibodies and Western blot2.6 Purification of Tm-proPO,Tm-SPE and Tm-SPH2 zymogen2.6.1 Preparation of E1,E2 and E3 fractions by using Toyopearl AF-Heparin HC column2.6.2 Purification of Tm-pro-PO protein from E2 fraction2.6.3 Purification of zymogenic and active form of 44 kDa proteins from E2 fraction2.6.3.1 Purification of zymogenic form of SPE2.6.3.2 Purification of active form of SPE2.6.4 Purification of Tm-SPH 1 protein2.6.5 Purification of Tm-SPH 2 protein from Toyopearl AF-Heparin HC column flow-through fraction2.7 Injection of protein into Tenebrio Molitor larvae2.8 Assay of antibacterial activity3.RESULTS3.1 The injection of active form of Tm-SPE to Tenebrio larvae induced melanin synthesis in vivo3.2 Tm-SPE can cleave Tm-proPO into three fractions3.3 Tm-SPE specifically cleaved Tm-SPH1 but not Tm-SPH23.4 Chemically cross-linked adducts are generated by the melanization complex3.5 Locally concentrated melanin on the surface of bacteria shows bactericidal effect4.DISCUSSION5.REFERENCES6.ACKNOWLEDGEMENT
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标签:黄粉虫论文; 先天性免疫论文; 酚氧化酶原论文; 黑色素论文; 酶级联系统论文; 细菌论文;
Molecular Regulation Mechanism of Phenoloxidase-induced Melanin Synthesis in the Mealworm Beetle, Tenebrio Molitor Larvae
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