家蚕模式识别受体PGRP、βGRP编码基因的克隆鉴定及表达谱分析

模式识别受体(PRR)是生物体先天免疫系统中免疫受体的代表,对生物体的生存极为重要。克隆鉴定了家蚕的14个模式识别受体编码基因,包括10个肽聚糖识别蛋白(PGRP)基因和4个β-葡聚糖识别蛋白(βGRP)基因,并得到了BmPGRP-S3、BmPGRP-S4和BmPGRP-L5的完整编码序列。家蚕PGRP的长型和短型亚家族都具有典型的酰胺酶活性结构域,短型亚家族具有信号肽,长型亚家族则没有信号肽。5个PGRP长型亚家族基因成簇分布于第1号染色体;5个PGRP短型亚家族基因中有2个分布于第9号染色体,有3个分布于第16号染色体。家蚕βGRP家族成员都具有信号肽,其中BmβGRP1-3成簇分布于第11号染色体,编码蛋白不具有典型的葡聚糖结合结构域;BmβGRP4独立分布于第22号染色体,编码蛋白具有典型的葡聚糖结合结构域。基因芯片数据分析表明,BmPGRP-L5和BmβGRP1在5龄第3天幼虫各组织中没有表达,其余12个模式识别受体基因为多组织表达,但在丝腺组织中均无表达。在这12个模式识别受体基因中,BmPGRP-L3等6个模式识别受体基因在中肠组织中的表达水平偏高;BmβGRP3、BmβGRP4和BmPGRP-L3、BmPGRP-L4等在家蚕生殖腺中的表达水平较高。在生殖腺以外的其他各组织中,这12个基因的表达不具有雌雄差异性。BmβGRP1在家蚕各发育时期没有表达,BmPGRP-L5主要在变态发育的转折期表达,其余12个模式识别受体基因在各发育时期均有表达,并从5龄第3天幼虫到上蔟第2天有较高水平的表达,雌雄个体间无表达差异性。由此说明这些模式识别受体基因的表达具有一定的组织性和发育时期性。给人工饲料无菌饲养的家蚕5龄第3天幼虫分别添食大肠杆菌、家蚕黑胸败血菌和家蚕白僵菌,对免疫诱导3、6、12和24h的家蚕进行个体水平的实时荧光定量PCR检测,发现PGRP长型亚家族基因BmPGRP-L1、BmPGRP-L3和短型亚家族基因BmPGRP-S1-3均能被这3种微生物诱导上调表达;βGRP基因家族中的BmβGRP3、BmβGRP4也能被3种微生物诱导上调表达。同时对诱导12h时家蚕各组织中14个家蚕模式识别受体基因的表达谱进行分析,结果显示经3种微生物分别诱导后,家蚕头部组织中BmβGRP2、BmβGRP4、BmPGRP-L2和BmPGRP-S1、BmPGRP-S3-5均上调表达;表皮、中肠和脂肪体中仅有BmβGRP3、BmPGRP-L4等少数模式识别受体基因上调表达。     

Bacterial cell wall components of Staphylococcus aureus induce endophthalmitis

AIM: To compare the responses of intraocular inflammation induced by heat-inactivated Staphylococcus aureus or its bacterial cell wall components in SD rats. METHODS: The rats were randomly divided into heat-inactivated bacteria (HIB) group (96 rats were injected with 10 μg of HIB), heat-inactivated bacteria fragments (HIBF) group (96 rats were injected with 10 μg of HIBF), peptidoglycan (PGN) group (96 rats were injected with 10 μg of PGN) and control group (96 rats were injected with sterile saline equivalent). At time points of 6 h, 12 h, 24 h, 48 h, 72 h and 5 d after vitreous injection of the pathogens, the ocular inflammation scores were determined under slit lamp microscope. The infiltration of white blood cells were counted in histological sections. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and cytokine induced neutrophil chemoattractant-1 (CINC-1) in serum and vitreous body were detected by ELISA, and protein concentration in aqueous humor were also measured. RESULTS: Severe ocular inflammation was observed in the animals of HIB, HIBF and PGN groups 6-72 h after injection. Five days after injection, the endophthalmitis subsided. The peak of intraocular white blood cell infiltration was observed 24 h after exposure to the bacteria and the components in each group and the cell infiltration rapidly declined after 3 days. The concentrations of TNF-α and IL-1β peaked at 24 h in each group, maintained to 48 h, then decreased rapidly, and returned to baseline level after 5 days. The concentration of CINC-1 peaked at 12 h in each group, and maintained to 24 h, then decreased rapidly, and returned to the normal level after 3 days. Significantly higher protein levels in aqueous humor were detected in the experimental groups at all time points as compared to that in control group (P<0.01). CONCLUSION: Staphylococcus aureus cells and its components induce typical experimental endophthalmitis in SD rats. Massive leukocyte infiltration and high levels of TNF-α, IL-1β and CINC-1 are the main pathological features in the experimental model. PGN and the bacterial cell wall fragments induce stronger intraocular inflammations than the whole heat-inactivated S. aureus.     

Digestion of microbial biomass, structural polysaccharides, and protein by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In order to investigate whether microbial biomass and its residues are nutrient and energy sources for humivorous beetle larvae, we carried out feeding trials using soil supplemented with ¹⁴C-labeled fungal biomass (Penicillium chrysogenum), bacterial biomass (Bacillus megaterium), fungal or bacterial structural polysaccharide (chitin, peptidoglycan), bacterial protein, or cellulose, taking the larva of the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model of a humus-feeding beetle larva with a highly alkaline gut. The results showed that gut passage strongly stimulated the mineralization of the structural polymers. The stimulatory effect correlated positively with the recalcitrance of the preparation in the control soil, and was accompanied by a transformation of the residual radiolabel to alkali-soluble and acid-soluble products. The solubility increase was highest in the extremely alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by reciprocal changes in the molecular weight of the residual material and that the residual material in the fecal pellets was more humified than in the control soil. The amount of radiolabel recovered from the body and hemolymph of the larvae indicated that microbial biomass and its structural components were assimilated more efficiently than cellulose, which supports the hypothesis that microorganisms and the nitrogenous components of humus are an important dietary resource for humivorous soil macroinvertebrates.     

肽聚糖对肉制品中产气荚膜梭菌芽孢萌发率影响及预测

该研究利用产气荚膜梭菌(Clostridium perfringens,C.perfringens)营养体及其芽孢肽聚糖以芽孢萌发率S、浑浊度OD600%、Ca^2+-DPA%变化率等为指标比较不同肽聚糖对C.perfringens芽孢萌发的影响;并针对芽孢萌发率检测耗时、费力等问题,提出一种基于近红外光谱技术(near infrared spectroscopy,NIR)定量预测不同浓度肽聚糖诱导芽孢萌发率研究。首先原始光谱经不同方式预处理,获得最佳方法为标准正态变换,然后使用主成分分析和遗传-联合区间偏最小二乘法进行光谱数据降维及特征变量筛选,分别对不同浓度肽聚糖诱导芽孢S、OD600%、Ca2+-DPA%进行快速预测。结果表明:营养体肽聚糖可有效诱导芽孢萌发,而芽孢肽聚糖效果不明显。利用GA-siPLS筛选芽孢萌发特征变量的最佳特征区间分别是[3,9,11,14]、[1,7,12,15]和[7,8,12,17],其预测集R和RMSEP分别为0.8726,0.8611,0.8841和0.769,0.218%,42.34%。研究结果表明,利用NIR结合GA-siPLS可定量预测肽聚糖诱导C.perfringens芽孢的萌发率,实现芽孢萌发的快速预测,为保证肉制品安全提供有效手段。     

皱纹盘鲍肽聚糖识别蛋白在免疫防御中的作用

本研究从皱纹盘鲍(Haliotis discus hannai)中鉴定并克隆了一种肽聚糖识别蛋白(PGRP),命名为HdPGRP。HdPGRP的cDNA全长为1467 bp,共编码354个氨基酸,其中含有1个信号肽(1~18氨基酸)、1个SH3b结构域(93~160氨基酸)、1个PGRP结构域(179~322氨基酸)和1个Ami_2结构域(191~332氨基酸)。此外,在HdPGRP序列中发现了4个保守的Zn2+结合位点(H209、Y255、H318和C330)以及5个保守的酰胺酶催化位点(H209、Y255、H318、T328和C330)。经多序列比对和系统发育树分析,表明HdPGRP属于短型PGRP家族成员。在健康鲍鱼中,hdpgrp主要在肝胰腺中表达,其次依次在血细胞、外套膜和鳃中。在鳗弧菌(Vibrio anguillarum)刺激后,血细胞中的hdpgrp表达量在72 h内呈现先上升后下降的趋势,在24 h表达量达到最高。SDS-PAGE结果显示,重组HdPGRP (rHdPGRP)的分子量为30 kDa。rHdPGRP表现为Zn2+依赖酰胺酶活性,可催化降解不溶性肽聚糖。此外,rHdPGRP对革兰氏阳性菌藤黄微球菌(Micrococcus luteus)具有显著的抑制作用,且这种抑制作用可能与其酰胺酶活性有关。本研究表明,HdPGRP在机体抵御入侵细菌等免疫防御中起重要作用。     

The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.     

免疫增强剂-肽聚糖在对虾养殖中的应用

概述肽聚糖的来源、结构组成，以及肽聚糖被利用后对提高生物多种免疫因子活力，增强抗病力所表现出的生物活性。分析了对虾免疫系统在防御病原入侵过程中，免疫细胞与体液免疫因子的功能特点，从对病原的防御，消除及损伤修复等方面阐明肽聚糖提高对虾非特异性免疫力的作用机理。     

Lb.casei.Zhang肽聚糖体内外抗肿瘤作用

为研究干酪乳杆菌Lb.casei.Zhang细胞壁肽聚糖(peptidoglycan;PG)的抗肿瘤作用,用流式细胞仪检测肽聚糖对人胃癌BGC-823细胞生长周期的影响。以肝癌H22细胞移植瘤昆明小鼠为模型,分别采用原位杂交和免疫组化方法检测小鼠移植瘤增殖细胞核抗原(PCNA)mRNA的表达以及凋亡因子Bcl-2和Bax基因的蛋白表达。结果显示,肽聚糖能够阻滞人胃癌BGC-823细胞生长于G0/G1期;实验组PCNA mRNA的表达和Bcl-2基因的蛋白表达平均光密度值均明显低于对照组,而Bax基因蛋白表达情况则相反,与对照组比较,均差异显著(α=0.05),且抑瘤率达37.21%。提示干酪乳杆菌Lb.casei.Zhang细胞壁肽聚糖具有抗肿瘤作用。     

昆虫先天免疫的肽聚糖识别蛋白的研究进展

先天免疫系统的激活是通过一系列高度保守的模式识别受体识别病原体相关分子模式。肽聚糖识别蛋白家族是重要的模式识别受体,从昆虫到人类均高度保守,可识别肽聚糖和含肽聚糖的细菌,在先天免疫和获得性免疫应答中发挥重要的识别和调节功能。     

Selective digestion of the peptide and polysaccharide components of synthetic humic acids by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In order to identify the potential nutrient and energy sources of humivorous beetle larvae, we carried out feeding trials with soil supplemented with specifically ¹⁴C-labeled model humic acids synthesized by peroxidase-initiated radical polymerization, using the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model organism. Ingestion of soil by the larvae significantly increased the mineralization of humic acids labeled in their peptide (HA-*peptide) or polysaccharide components (HA-*peptidoglycan and HA-*chitin), whereas the mineralization of humic acids labeled in the aromatic components (HA-*catechol) did not increase significantly. Mineralization was accompanied by a reduction of residual radiolabel in the acid-soluble fraction and an increase in the humic acid and humin fractions of the fecal pellets. During the gut passage, the residual label in peptide or polysaccharide components was transformed into acid-soluble products, especially in the alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by large changes in the molecular weight of the residual material. The amount of radiolabel derived from the peptide and polysaccharide components recovered from the larval body and hemolymph was significantly higher than that derived from the aromatic component, which supports the hypothesis that humivorous beetle larvae selectively digest the peptide and polysaccharide components of humic substances, whereas the aromatic components of humic substances are not an important source of nutrients and energy. This is also the first experimental evidence that also chitin and peptidoglycan, the major structural polymers in fungal and bacterial biomass, can be protected from microbial degradation in soil by a copolymerization with phenols and might contribute substantially to the refractory nitrogen pool in soil organic matter.