长双歧杆菌完整肽聚糖对小鼠免疫功能的影响

目的比较长双歧杆菌及其完整肽聚糖的免疫调节作用。方法通过研究长双歧杆菌完整肽聚糖对植瘤小鼠淋巴细胞的转化、抑瘤率、生命延长期以及对细胞Bcl-2和Bax表达的影响来探讨它们对小鼠细胞免疫的调节作用。结果与对照组相比,完整肽聚糖使淋巴细胞转化增加、抑瘤率提高、延长生命期增加、Bcl-2阳性表达率减小而Bax基因表达增加。结论长双歧杆菌与其完整肽聚糖均有抑制S180肿瘤的作用,但完整肽聚糖的效果优于长双歧杆菌。     

双歧双歧杆菌细胞壁完整肽聚糖的分离纯化

本试验采用国内分离的双歧双歧杆菌菌株Ｂｉｆ１１０１,用ＴｒｉｔｏｎＸ－１００处理ＢＬ肉汤培养物,高速离心分离不可溶沉渣,经蛋白酶Ｅ、胃蛋白酶、α－糜蛋白酶、胰酶、核酸酶消化,甲醇与氯仿除脂,０．０１ＮＨ２ＳＯ４处理等步骤提纯而得到精制完整肽聚糖（ｗｈｏｌｅｐｅｐｔｉｄｏｇｌｙｃａｎ,ＷＰＧ）。化学组分分析结果显示ＷＰＧ主要由多肽与糖组成,其氨基酸组成与菌体基本相同。扫描电镜观察发现提取的ＷＰＧ保留其原菌体细胞完整结构。     

双歧杆菌的完整肽聚糖对巨噬细胞NF-kB的影响

目的从NF-kb角度探索分叉双歧杆菌的完整肽聚糖（WPG）激活巨噬细胞的信号机制.方法首先分离培养SD大鼠腹腔巨噬细胞,然后以不同浓度的WPG刺激巨噬细胞,最后采用凝胶电泳迁移分析技术检测巨噬细胞NF-kb的DNA结合活性.结果分叉双歧杆菌的WPG作用于巨噬细胞后,其NF-kB的DNA结合活性明显高于对照组（P＜0.01）.结论分叉双歧杆菌的WPG能活化巨噬细胞的NF-kb.     

双歧杆菌肽聚糖结构及分子量的分析

从双歧杆菌细胞壁中分离纯化肽聚糖,研究其化学成分、结构和分子量分析。经氨基酸、多糖和蛋白质含量分析和肽聚糖的溶解实验鉴定,所提取物质的主要成分为肽聚糖;经核磁共振和红外光谱分析,所提取肽聚糖的结构与已知的肽聚糖的结构相吻合,其糖链结构为由D型吡喃糖N-乙酰葡糖胺和N-乙酰胞壁酸以β-1,4-糖苷键连接而组成;经SDS-PAGE和多种染色方法结合,分析经溶菌酶水解后的肽聚糖,其分子量的分布呈弥散状,范围在97.6kD到14.4kD之间。     

双歧杆菌的完整肽聚糖对实验性大肠癌凋亡控制基因表达的影响

为探讨双歧杆菌的完整肽聚糖的抑瘤途径及机制,本文以大肠癌裸鼠移植瘤为动物模型,采用免疫组化ＳＰ法检测了４０只裸鼠移植瘤ｂｃｌ－２及ｂａｘ基因的蛋白表达率及表达强度,结果显示肽聚糖注射组大肠癌移植瘤ｂｃｌ－２蛋白表达率及阳性细胞密度低于肿瘤对照组,ｂｃｘ基因的表达情况则相反。提示双歧杆菌的完整肽聚糖可使大肠癌裸鼠移植瘤的ｂｃｌ－２基因表达下调,ｂａｘ基因表达强,最终诱导肿瘤细胞凋亡,实现其抗瘤目的。     

双歧杆菌的完整肽聚糖对巨噬细胞AP-1的影响

目的探索信号途径ERK→AP-1在双歧杆菌的完整肽聚糖(WPG)激活巨噬细胞中的作用。方法以WPG刺激SD大鼠腹腔巨噬细胞,采用凝胶电泳迁移分析技术检测巨噬细胞AP-1的活性。结果WPG刺激组巨噬细胞AP-1的活性明显高于对照组(P<0.01);巨噬细胞经ERK抑制剂PD98059预孵后,其AP-1的活性显著低于WPG刺激组(P<0.01)。结论双歧杆菌的WPG可通过ERK→AP-1这一信号途径来活化巨噬细胞。     

双歧杆菌的完整肽聚糖对大鼠腹腔巨噬细胞TNF-α表达的影响

目的探索双歧双歧杆菌的完整肽聚糖(WPG)对大鼠腹腔巨噬细胞TNF-α的调控作用。方法以WPG刺激大鼠腹腔巨噬细胞;或以NF-κB的抑制剂NAC和ERK抑制剂PD98059分别预先孵育巨噬细胞,再用WPG刺激巨噬细胞,最后用RT-PCR检测巨噬细胞TNF-αmRNA的表达水平。结果 WPG刺激巨噬细胞后,其TNF-αmRNA的表达水平显著高于对照组(P0.01)。但经NAC和PD98059分别预先孵育巨噬细胞后,其TNF-αmRNA的表达水平均明显低于WPG刺激组(P0.01)。结论双歧双歧杆菌的WPG能上调巨噬细胞TNF-αmRNA的表达,这一过程受NF-κB和ERK的调控。     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞细胞骨架的影响

目的探讨双歧双歧杆菌的完整肽聚糖（WPG）对巨噬细胞细胞骨架的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞后,用荧光标记的鬼笔环肽染液染色,最后采用激光共聚焦显微镜技术检测巨噬细胞的细胞骨架。结果和对照组相比,WPG刺激巨噬细胞后,其胞内肌动蛋白减少且排列更加紊乱,同时胞膜荧光强度减弱,胞外放射状荧光物质减少,甚至消失。结论双歧双歧杆菌的WPG在激活巨噬细胞的过程中可影响其细胞骨架。     

双歧杆菌的完整肽聚糖对实验性大肠癌凋亡调控基因表达的影响

为探讨双歧杆菌的完整肽聚糖的抑瘤途径及机制,本文以大肠癌裸鼠移植瘤为动物模型,采用免疫组化ＳＰ法检测了４０只裸鼠移植瘤ｂｃｌ－２及ｂａｘ基因的蛋白表达率及表达强度。结果显示完整肽聚糖注射组大肠癌移植瘤ｂｃｌ－２蛋白表达率及阳性细胞密度均低于肿瘤对照组,ｂａｘ基因的表达情况则相反。提示双歧杆菌的完整肽聚糖可使大肠癌裸鼠移植瘤的ｂｃｌ－２基因表达下调,ｂａｘ基因表达增强,最终诱导肿瘤细胞凋亡,实现其抗瘤目的。     

双歧杆菌的完整肽聚糖对实验性大肠癌诱导型一氧化氮合酶表达的影响

建立大肠癌裸鼠移植瘤模型,以免疫组化法观察大肠癌移植瘤一氧化氮合酶（iNOS）基因的蛋白表达水平,结果显示完整肽聚糖（Whole peptidoglycan,WPG）注射组大肠癌移植瘤iNOS蛋白的表达率、表达强度以及阳性细胞密度均明显高于肿瘤对照组（P＜0.01）,提示分叉双歧杆菌的WPG能增强大肠癌移植瘤细胞iNOS基因的蛋白表达,这可能是其抑瘤机制之一。     

Linear and Conformation-Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.     

狂犬病毒核蛋白（NP）的纯化及其免疫原性的研究

对重组痘苗毒和重杆状病毒表达的狂犬病毒NP及原代地鼠肾细胞培养的狂犬病毒核衣壳蛋白（RNP）、先经Sepharose CL 4B分子筛柱初步提纯,再经抗狂犬病毒NP McAB 2C12-S-epharose 4B亲和层析柱纯化分离,经ELISA、SDS－PAGE电泳和Western-Blot分析证实,获得了高纯度和免疫反应性的NP和RNP。以相同剂量的纯化蛋白免疫小鼠,RNP和两种重组NP均可诱生特异的抗NP抗体,三种蛋白间无明显差异;狂犬病毒CVS株攻击保护实验结果显示,三种蛋白免疫的小鼠存活率约为50％;两种重组NP的免疫反应性和免疫原性与天然狂犬病毒RNP相似。     

Morphology and Antigenic Properties of Recombinant Analogs of the Marburg Virus Nucleoprotein

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the T7 RNA polymerase promoter. Recombinant NP was synthesized in Escherichia coliand in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coliand of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.     

miR-769-3p 对甲型 H1N1流感病毒核蛋白表达的调控

目的:预测靶向甲型流感病毒核蛋白(NP)基的微小 RNA(miRNA),并检测其对 NP 表达的影响.方法:从miRBase 数据库中获取人成熟 miRNA 序列,利用 miRanda 软件预测潜在靶向流感病毒 A/FM/1/47(H1N1) NP 基的人 miRNA;通过双萤光素酶报告基系统及 Western 印迹验证所预测的 miRNA 对 NP 表达的影响.结果:用 miRanda软件在流感病毒 A/FM/1/47(H1N1) NP 基上预测得到分值及最小结合自由能均较好的 miR-769-3p;双萤光素酶报告基结果显示 miR-769-3p 能显著降低报告基载体萤光素酶的表达;Western 印迹结果显示 miR-769-3p 能明显抑制 NP 的表达,但突变 NP 基上的 miR-769-3p 结合位点后,miR-769-3p 不能抑制 NP 的表达.结论:miR-769-3p 可靶向流感病毒 A/FM/1/47(H1N1) NP 基并抑制 NP 的表达,为抗甲型流感病毒的 miRNA 药物研发提供了据和潜在药物靶标.     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及ＨＳＰ７０的表达与病毒复制的关系,在汉滩病毒Ａ９株感染Ｖｅｒｏ－Ｅ６细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞ＨＳＰ７０基因的表达进行了检测。结果表明,汉滩病毒感染细胞４ｈｙ后即可诱导Ｖｅｒｐ－Ｅ６细胞表达ＨＳＰ７０,表达可持续至感染后５ｄ且ＨＳＰ７０在细胞内的分布也有改变。提示汉滩病毒可直接诱导ＨＳＰ７０的高表达。     

白细胞介素-2和狂犬病毒N蛋白基因重组与表达

研究白细胞介素－２ 与狂犬病毒Ｎ蛋白基因工程产物的免疫活性。采用生物工程技术,结果显示经基因扩增分别获得４００ｂｐ 和１４００ｂｐ 的ＩＬ－２ 和狂犬病毒Ｎ蛋白基因片段,构建了表达载体ＰＬＹ－４ＩＮ,于工程菌中转化后。得到５６ＫＤ重组蛋白产物,该产物具有ＩＬ－２活性,可诱导小鼠抵抗狂犬病毒攻击。     

戊型肝炎病毒重组颗粒性蛋白疫苗在小鼠体内诱导的免疫应答研究

HEV 239是福建省医学分子病毒学研究中心实验室研制的一种戊型肝炎病毒（HEV）重组颗粒性蛋白疫苗,该文旨在研究HEV239蛋白疫苗在小鼠体内诱导产生特异性免疫应答的情况.将5μg HEV 239蛋白疫苗（239-Pro）、加铝佐剂疫苗（239-Vac）或加弗氏佐剂疫苗（239-CFA）肌肉注射免疫BALB/c鼠3次,第8周检测鼠血清抗HEV抗体及其亚类,同时用ELISPOT方法检测细胞毒性T细胞（CTL）应答.结果显示：239-Vac诱导的抗体滴度与239-CFA相当,高于无佐剂的239-Pro.239-Vac诱导的抗体中,IgG1/IgG2a比值显著高于239-CFA和239-Pro,主要为Th2型应答.除239-CFA之外,239-Vac和239-Pro也可诱导出一定的HEV抗原特异性I型Tc应答.提示：重组抗原HEV 239能诱导良好的抗体应答及一定的Tc1应答.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).