重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

旨在获得产气荚膜梭菌β毒素（CPB）的重组突变体,并评价其毒力及免疫保护性。对已知的产气荚膜梭菌CPB编码基因进行优化设计,同时引入4个氨基酸突变位点,分别是第212位的精氨酸突变为谷氨酸,第268位的亮氨酸突变为甘氨酸,266位的酪氨酸和275位的色氨酸突变为丙氨酸。此外,在该基因5'端添加Th细胞表位（T）和鞭毛蛋白（flagellin）N末端的编码序列,经人工合成获得重组基因片段（GTF_NCPB_m4）。将GTF_NCPB_m4克隆至原核表达载体pET-30a（＋）中进行表达与纯化,获得重组蛋白rTF_NCPB_m4。利用Western blot方法检测rTF_NCPB_m4与C型产气荚膜梭菌毒素抗血清的反应性,并检测rTF_NCPB_m4对小鼠的毒力。随后,以rTF_NCPB_m4免疫家兔,按照《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清对C型产气荚膜梭菌毒素的中和抗体效价。结果表明,rTF_NCPB_m4主要以包涵体的形式表达且能与C型产气荚膜梭菌毒素抗血清反应。小鼠安全性试验显示,50 μg的rTF_NCPB_m4对小鼠仍无致死性;免疫rTF_NCPB_m4后,每毫升家兔二免抗血清可中和10~20个小鼠最小致死量（MLD）的C型产气荚膜梭菌毒素;1个家兔MLD的C型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%（4/4）的保护。以上结果说明,rTF_NCPB_m4在丧失毒力的同时保留了良好的免疫原性,从而为C型产气荚膜梭菌病基因工程疫苗的研制提供了重要的数据。     

金黄色葡萄球菌CP5偶联鞭毛蛋白疫苗对小鼠乳腺炎模型的保护作用

以鞭毛蛋白(flagellin)作为载体蛋白,通过化学偶联至金黄色葡萄球菌5型荚膜多糖(CP5),并在小鼠模型上检验多糖疫苗对小鼠存活及其乳腺的保护作用.将小鼠分为4组:生理盐水组、CP5单独免疫组、牛血清白蛋白(BSA)-CP5偶联组和flagellinCP5偶联组,每组10只,分别进行皮下免疫,均含CP5 10 μg;共免疫两次,两次免疫间隔3周.第2次免疫两周后,用金黄色葡萄球菌分别对小鼠腹腔、乳腺进行攻毒,并通过实时定量PCR对乳腺组织细胞因子IL-4、IL-10、IFN-γ、IL-12的表达进行检测.结果表明,CP5偶联鞭毛蛋白疫苗对小鼠乳腺具有显著的保护效果,并在乳腺组织部位诱导了Th1型细胞因子表达,说明鞭毛蛋白是CP5的理想载体蛋白.     

猪源大肠杆菌O157:H7鞭毛蛋白抗原表位分析及验证

利用生物信息学分析大肠杆菌O157:H7鞭毛蛋白FliC的二级结构及亲水性、抗原指数、柔性区域和表面可能性等指数,预测大肠杆菌O157:H7鞭毛蛋白FliC的潜在B细胞抗原表位,为其致病性研究提供理论基础。利用DNAStar软件Protean程序中Garnier-Robson方法和Chou-Fasman方法分析鞭毛蛋白FliC的α-螺旋、β-折叠、转角区域和卷曲区域,通过Kyte-Doolittle方法、Karplus-Schulz方法、Emini方法和Jameson-Wolf方法分析鞭毛蛋白FliC的亲水性、柔性区域、表面可能性和抗原指数。综合分析得出鞭毛蛋白FliC 63-74、236-247、338-349、460-471、542-553位氨基酸序列为潜在的B细胞优势抗原表位。化学合成法合成优势抗原表位338-349和460-471肽段,免疫BALB/c小鼠3次后,采用ELISA方法验证抗体水平。ELISA结果显示,338-349、460-471肽段具有很强的抗原性,能引起BALB/c小鼠产生高滴度的抗体。     

Molecular cloning and functional identification of an apple flagellin receptor MdFLS2 gene

The leucine-rich repeat receptor kinase flagellin-sensing 2 gene(MdFLS2; Gene ID: MDP0000254112) was cloned from Royal Gala apple(Malus×domestica Borkh.). This gene contained a complete open reading frame of 3 474 bp that encoded 1 158 amino acids. The phylogenetic tree indicated that Prunus persica FLS2 exhibited the highest sequence similarity to MdFLS2. The PlantCare database suggests that the promoter sequence of MdFLS2 contains several typical cis-acting elements, including ethylene-, gibberellin-, salicylic acid-, and drought-responsive elements. Quantitative real-time PCR analysis showed that MdFLS2 was widely expressed in the different tissues of the apple and most highly expressed in the leaves. Furthermore, MdFLS2 was significantly induced by the flagellin elicitor peptide flg22. Treatment of the apple seedling leaves with flg22 resulted in an increase in leaf callose levels with increased treatment duration. An increase in the production of O~(2–) along with the expression of disease-related genes was also observed. An oxidative burst was detected in the treated seedlings, but not in the control seedlings, indicating that flg22 had stimulated the expression of the MdFLS2 gene and its downstream target genes. Furthermore, the ectopic expression of MdFLS2 complemented the function of the Arabidopsis fls2 mutant and conferred enhanced flg22 tolerance to the transgenic Arabidopsis, suggesting that MdFLS2 acts as a positive regulator in the response to pathogens in apple.     

细菌鞭毛素对植物免疫防卫反应及其信号机制的激发

细菌鞭毛素(flagellin)是一类可以诱导寄主植物细胞发生防卫反应的病原物相关分子模式(PAMP)因子,其活性位点是被称为flg22的N 末端保守的22个氨基酸多肽,植物体内与其结合的模式识别受体是FLS2。flg22可以诱导植物细胞发生活性氧(ROS)的猝发、细胞培养介质碱化、一氧化氮(NO)的产生,胼胝质沉积以及MAPK级联反应、防卫基因表达等多种防卫反应。本文结合本室有关水稻白叶枯病菌的研究结果,综述近年来国内外在鞭毛素诱导植物防卫反应研究领域的最新研究进展。     

贝莱斯芽孢杆菌LJ02中植物免疫蛋白的筛选及其功能

【目的】贝莱斯芽孢杆菌（Bacillus velezensis）LJ02菌株能够诱导黄瓜等植物产生免疫抗病反应。本研究从生防菌LJ02中分离鉴定具有激发植物免疫活性的蛋白分子,通过验证其功能,进一步解析免疫蛋白的免疫信号通路。【方法】用硫酸铵沉淀法处理LJ02菌株发酵液,离心获得LJ02蛋白粗提物,将其经凝胶层析后再利用半制备高效液相色谱（high performance liquid chromatography,HPLC）分离,收集不同峰值处的蛋白组分。以烟草花叶病毒（tobacco mosaic virus,TMV）为靶标进行活性组分的筛选,从而获得具有免疫活性的组分。液相质谱联用仪（LC-MS）检测分析活性组分后发现,在组分F-23中含鞭毛蛋白（flagellin,Flg^LJ02）。将鞭毛蛋白Flg^LJ02进行原核表达并纯化,经过敏性坏死反应（hypersensitive reaction,HR）和抗病试验验证其免疫功能。为确定其主要免疫信号通路,采用实时荧光定量PCR（qRT-PCR）检测水杨酸（salicylic acid,SA）和乙烯（ethylene,ET）途径的相关合成酶基因ICS1、PAL、ACS1以及免疫相关抗性基因NPR1、PR-1、EIN2和EIN3,通过免疫相关抗性基因的相对表达量解析鞭毛蛋白Flg^LJ02免疫信号转导途径。【结果】HPLC层析分离出贝莱斯芽孢杆菌LJ02菌液产生的60个外泌蛋白组分（F-1—F-60）,免疫烟草接种TMV后,观察分析发现组分F-20、F-23、F-41、F-44具有显著的免疫抗病效应,其中组分F-23抗TMV效果最为显著,其抑制率为81.7%。经过质谱数据分析发现该组分包含鞭毛蛋白Flg^LJ02等7种物质。选择鞭毛蛋白Flg^LJ02为研究对象构建表达载体,转化大肠杆菌BL21,诱导表达后破碎细胞,获得粗提蛋白。将粗提蛋白过柱、透析纯化后的Flg^LJ02渗入珊西烟（Nicotiana tabacum var. xanthi）叶片,24 h后能观察到过敏性坏死反应。将Flg^LJ02稀释为终浓度50、100、200 μg·mL ^-1的稀释液后预处理珊西烟叶片,24 h后接种TMV,其对TMV的枯斑抑制率分别为65.6%、76.1%、88.1%。用鞭毛蛋白Flg^LJ02处理珊西烟,其SA途径和ET信号途径的免疫抗病相关基因ICS1、PAL、NPR1、PR-1、ACS1、EIN2、EIN3的相对表达量在24—48 h均显著上调。【结论】贝莱斯芽孢杆菌LJ02外泌的鞭毛蛋白Flg^LJ02可以激发珊西烟植株的免疫防御反应,提高烟草植株对TMV的抗病性。     

Protective effect of MUC2 in mice with colitis and the expression of anti-CBir1 antibody in serum

AIM: To investigate the protective effect of mucin 2 (MUC2) on intestinal mucosa of colitis model mice, and to explore the correlation between the expression of anti-CBir1 flagellin antibody and MUC2. METHODS: The mice were randomly divided into normal control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS) group, lipopolysaccharide (LPS)+ovalalbumin (OVA)+TNBS group and ketotifen+TNBS group. The expression of MUC2 in colon tissue was determined by PAS staining and immunohistochemistry, and the anti-CBir1 antibody level in the serum of mice in each group was measured by ELISA. RESULTS: The scores of disease activity index and histological index in TNBS group were higher than those in normal control group (P<0.05). The scores in LPS+OVA+TNBS group were much higher than those in TNBS group (P<0.05). However, the values in ketotifen+TNBS group were lower than those in TNBS group (P<0.05). PAS staining showed a decrease in goblet cells in TNBS group. Compared with TNBS group, the colonic mucosa integrity in LPS+OVA+TNBS group was destroyed, and the number of goblet cells in ketotifen+TNBS group increased significantly. Immunohistochemical staining showed that the expression of MUC2 in the intestinal tract of each mo-del group was basically consistent with the results of PAS staining. The serum anti-CBir1 antibody level in TNBS group was higher than that in normal control group (P<0.05), and that in LPS+OVA+TNBS group was significantly higher than that in TNBS group (P<0.05), whereas that in ketotifen+TNBS group was decreased slightly (P<0.05). CONCLUSION: MUC2 plays a protective role in the pathogenesis of colitis in mice, and there is a negative correlation between the expression of MUC2 and the bacterial flagellin in the intestinal mucosa of mice with colitis.     

5种细菌鞭毛素蛋白诱导细胞因子IL-4、IFN-γ mRNA的表达水平

选择5种不同细菌来源的鞭毛素蛋白刺激猪外周血淋巴细胞,利用荧光定量PCR检测相关细胞因子白介素4(IL-4)和γ-干扰素(IFN-γ)mRNA的表达水平,进一步选择4个含量(0.1、1.0、5.0、10.0μg·m L~(-1))的创伤弧菌鞭毛素蛋白,探讨其诱导IFN-γ的表达效果.结果表明,鞭毛素蛋白可以显著提高IFN-γmRNA的表达水平(P0.05),以创伤弧菌(Vibrio vulnificus MO6-24/O)的效果最佳.4个含量的创伤弧菌鞭毛素蛋白均能显著提高猪外周血淋巴细胞IFN-γmRNA的表达量(P0.05),其中以5.0μg·m L~(-1)为创伤弧菌鞭毛素蛋白的最适含量.本试验主要目的是筛选一种能够有效提高猪免疫功能的细菌鞭毛素蛋白,为挖掘新型疫苗佐剂提供基础.     

空肠弯曲杆菌鞭毛的研究进展

空肠弯曲杆菌是一种人畜共患的食物源性病原菌，可引起人和动物细菌性腹泻，且该菌的感染率逐年递增。鞭毛是细菌菌体的一种特殊结构，与菌体的运动性密切相关，有助于其躲避有害环境，同时鞭毛在细菌的致病性等方面也起着重要作用。研究发现，空肠弯曲杆菌的发病机制与鞭毛在宿主上皮细胞的定植力、黏附、侵袭力及毒素的产生密切相关。文章概述了空肠弯曲杆菌鞭毛结构、功能、调控机制及相关基因等方面的研究进展，通过归纳总结已知基因缺失突变对鞭毛的影响，从分子水平了解鞭毛的调控机制，从而探讨空肠弯曲杆菌的致病机理，以期为降低其感染率提供理论依据。