【作者】 石怀兴；

【导师】 邱德文；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 致病杆菌（Xenorhabdus）是昆虫病原斯氏线虫（Steinernema）的肠道共生菌，能够产生多种毒素物质干扰宿主昆虫的免疫反应，协助昆虫病原线虫的成功侵染。本研究从长尾斯氏线虫（S.longicaudum）的共生致病杆菌（X. ehlersii）中分离纯化出一种具有注射活性的毒蛋白，该蛋白能够干扰大蜡螟（Galleria mellonella）的细胞免疫和体液免疫反应；克隆得到了其编码基因并进行了原核表达，用差异双向电泳和荧光定量PCR技术检测了该蛋白对大蜡螟蛋白组和基因转录本水平的影响。本研究为进一步揭示昆虫病原线虫-细菌共生体与宿主昆虫的相互作用关系奠定了基础。主要研究成果如下：1.分离纯化并鉴定了一种具有注射活性的毒蛋白。利用硫酸铵沉淀、HiTrap Q HP阴离子交换层析、HiTrap Butyl FF疏水层析和RESOURCE Q阴离子交换层析，从X. ehlersii胞内组分中分离得到一种具有注射活性的毒蛋白，双向电泳显示该蛋白表观分子~58kDa，等电点~5.0。注射该蛋白10min后大蜡螟全身黑化，并在48h内死亡，致死中量为0.76±0.08μg/头。该毒性蛋白经离子淌度质谱（HDMS）鉴定为一种GroEL同源蛋白，与来自嗜线虫致病杆菌（X. nematophila）的一种GroEL蛋白（XnGroEL）匹配度最高，将其命名为XeGroEL。2.检测了XeGroEL蛋白对大蜡螟细胞免疫及多酚氧化酶（PO）活性的影响。注射XeGroEL蛋白后，大蜡螟血细胞尤其是浆血细胞的数量显著减少，体外孵育不会显著降低血细胞数量，但能够使浆血细胞显著减少；体内注射和体外孵育均能减弱大蜡螟血细胞对琼脂糖小球的包被能力，而且经XeGroEL体外孵育的大蜡螟血细胞对荧光微球的吞噬率有所提高。XeGroEL蛋白能够显著提高大蜡螟血淋巴中的PO活性，但体内注射活性是体外孵育的5倍。3.克隆得到XeGroEL基因、进行了原核表达及抗体的制备。根据质谱鉴定结果，设计兼并引物，利用PCR克隆得到了XeGroEL基因，该基因全长1647bp，编码548个氨基酸，是一种在细菌中广泛存在的高度保守的看家基因，系统发育树分析表明其可作为一种辅助的分子标记用于肠杆菌科细菌的进化分析；共生菌和非共生菌的GroEL氨基酸序列分析显示，共生菌的GroEL保守性比非共生菌的差，GroEL在共生菌中的功能多样性可能与某些位点的不保守性有关。XeGroEL和XnGroEL的序列比对分析显示，二者活性的差异可能与其序列中17个氨基酸的置换而造成的空间结构差异有关。此外，利用pET28a载体，我们将XeGroEL在大肠杆菌中成功表达，纯化了重组XeGroEL并制备了多克隆抗体。4.检测了XeGroEL蛋白对大蜡螟蛋白质组和基因转录本水平的影响。差异双向电泳和荧光定量PCR显示，XeGroEL蛋白能够作为一种有效的免疫原物质刺激大蜡螟免疫相关因子的表达，注射XeGroEL蛋白后，大蜡螟体内的PGRP蛋白（PGRP-LB，PGRP-A和PGRP-B）、抗菌肽（阳离子抗菌肽，gallerimycin，cecropin和gloverin）、解毒蛋白（铁蛋白和GST）、17kDa的血淋巴蛋白和载脂蛋白-3的表达量出现不同程度的上调。此外，免疫共沉淀实验显示，XeGroEL蛋白能够和大蜡螟血淋巴中的两种蛋白结合，这为进一步研究其互作分子提供了基础。更多还原

【Abstract】 The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxicproteins that interfere the immune system of insects, which facilitate the successful infection ofentomopathogenic nematode Steinernema. We purified a toxic protein with injectable activity fromXenorhabdus ehlersii, and the toxic protein can interferet the cellular and humoral immune response inGalleria mellonella；we cloned the gene and expressed it in Escherichia coli. We determined the effectof XeGroEL on proteome and gene transcriptal level by comparative proteomic analyses andRealtime-qPCR. Our results help to understand anti-microbial immune responses in G. mellonella,providing a meaningful hypothesis for the interaction between nematode-symbiotic bacteria and host.The main results are as summarized as follows:1. A toxic protein with injectable activity was purified and identified.A toxic protein from X. ehlersii was purified by ammonium sulfate precipitation, sequential HiTrap QHP, HiTrap Butyl FF and RESOURCE Q chromatography, and identified as a GroEL homology with ahighest matching against a GroEL from X. nematophila by LC-MS/MS on HDMS High DefinitionMass Spectrometry system. The apparent molecular weight and isoelectric point were estimated as58kDa and5.0by2-D PAGE. G. mellonella larva injected with XeGroEL presented melanization after10min, and dead in48h. An LD50of0.76±0.08μg/larva was calculated.2. Effect of XeGroEL on hemocytes immune and PO activity in G. mellonella was determined.After injecting XeGroEL into the G. mellonella hemolymph, the total hemocyte number decreased,especially the number of plasmatocytes, and a minor reduction in hemocyte numbers was observed byincubation in vitro, however plasmatocytes were greatly diminished in both treatments. XeGroEL canweaken the encapsulation of G. mellonella hemocytes by injection or incubation in vitro, whilepromoted the phagocytosis of G. mellonella hemocytes in vitro. XeGroEL increased the PO activity inhemolymph，and the effect was more intensive in vivo.3. XeGroEL gene was cloned and then expressed in E. coli, and the polyclonal antibody againstrecombinant XeGroEL was prepared.According to the MS result, degenerate primers were designed for PCR, XeGroEL with1647bpencoding548amino acid residues is a ubiquitous house keeping geng in bacteria, phylogenetic analysisshowed that XeGroEL is a valuable molecular marker in evolution analysis of enterobacteriaceae;sequence anlignment showed that GroEL of symbiotics had lower conservation than that of free-livingbacteria, and the multifunctionality of GroEL in symbiotics may be related to several unconservativeposition. Alignment of XeGroEL and XnGroEL indicates that substitution at17positions may attributedto the different activity. In addition, we expressed the XeGroEL ligating to pET28a vector, andpolyclonal antibody was prepared using purified recombinant XeGroEL.4. The changes of proteome and transcriptal level in XeGroEL-challenged G. mellonella weredeteimined.we demonstrated that XeGroEL can promote the expression of some immune-related factors acting as an effective immunogen by comparative proteomic analyses and Realtime-qPCR. The expressionlevel of PGRPs (PGRP-LB, PGRP-A and PGRP-B), antimicrobial peptides (anionic antimicrobialpeptide2, gallerimycin, cecropin, gloverin), antidotic proteins (ferritin and GST),27kDa hemolymphprotein and apolipophorin-3in XeGroEL-challenged larvae were all increased. Additionally,co-immunoprecipitation presented2hemolymph proteins binding with XeGroEL, which provided thebasis for further interaction study.更多还原