结核病新疫苗的免疫毒理学评价方法研究

目的 建立疫苗评价用豚鼠免疫毒理学评价方法,并应用建立的评价指标对结核病新疫苗（耻垢疫苗）的免疫毒性进行初步评价。 方法 制备豚鼠过敏模型30只,阴性对照15只。建立豚鼠血清总IgG1抗体检测和外周血嗜碱粒细胞体外刺激培养上清、支气管肺泡灌洗液上清,以及腹腔灌洗液中肥大细胞体外刺激培养上清中的过敏性介质（组胺和白三烯）含量的检测方法。将豚鼠分成3组,分别为过敏模型组、耻垢疫苗组和阴性对照组,每组6只,应用已建立的以上几种评价指标初步进行了结核病新疫苗（耻垢疫苗）的免疫毒性评价。结果 以6 μg/ml羊抗豚鼠IgG1抗体包被,待测血清稀释1:10⁵,以及辣根过氧化物酶(HRP)标记的羊抗豚鼠IgG1抗体稀释1:20 000的实验条件为豚鼠血清总IgG1抗体的最佳检测方法。外周血嗜碱粒细胞体外刺激培养上清中,豚鼠过敏模型组和对照组中组胺含量分别为（99.37±15.34）nmol/L和（29.94±5.86）nmol/L;白三烯含量分别为（13.09±3.87）pg/ml和（2.22±0.53）pg/ml。支气管肺泡灌洗液上清组胺含量分别为（73.42±18.60）nmol/L和（31.00±12.09）nmol/L,白三烯含量分别为（123.20±16.57）pg/ml和（39.05±16.94）pg/ml。腹腔灌洗液中肥大细胞体外刺激培养上清中组胺含量分别为（39.59±12.94）nmol/L和（14.35±5.85）nmol/L,白三烯含量分别为（6.79±2.58）pg/ml和（3.09±1.18）pg/ml。以上3种不同组织来源的样本中,过敏模型组的组胺和白三烯含量与对照组相比差异均有统计学意义（组胺：t=12.60, t=5.41, t=5.20,P＜0.05;白三烯：t=8.46, t=9.15, t=3.85,P＜0.05）。应用以上方法评价结核病新疫苗未见免疫学毒理学异常反应。结论 实验初步建立了免疫毒理学评价方法,以该方法评价耻垢疫苗未见免疫毒理学异常反应。     

结核分枝杆菌Rv0901基因重组耻垢分枝杆菌的构建及其细胞作用

目的对结核分枝杆菌Rv0901基因的功能进行研究。方法以PCR扩增Rv0901基因编码序列,定向克隆人穿梭表达质粒pMV261获得重组穿梭表达质粒,将重组质粒电穿孔进入耻垢分枝杆菌,构建重组Rv0901基因的耻垢分枝杆菌,对重组耻垢分枝杆菌进行诱导表达,用SDS-PAGE检测表达结果。比较耻垢分枝杆菌和重组耻垢分枝杆菌对THP-1细胞的不同作用。结果成功构建重组穿梭表达质粒及重组Rv0901基因的耻垢分枝杆菌,重组菌诱导THP-1细胞的凋亡率高于耻垢分枝杆菌,其感染THP-1细胞后细胞的存活率低于耻垢分枝杆菌的感染,重组菌感染THP-1细胞后细胞培养液中NO(一氧化氮)的产生高于耻垢分枝杆菌的感染。结论Rv0901基因可能与结核毒力存在一定关系。     

Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Compartmentalization is used by cells to overcome many difficult metabolic and physiological challenges (1). Eukaryotes employ membrane-bound organelles such as the mitochondrion (2); however, most prokaryotes rely on alternative proteinaceous compartments to achieve spatial control (3), one of which is the encapsulin nanocompartment.Encapsulins are newly identified nanocompartments but have already been applied in various scientific fields due to the unique structures (4, 5). It has been reported that more than 900 putative encapsulin systems in bacteria and archaea exist and are distributed across 15 bacterial and two archaeal phyla (6, 7), suggesting they are functionally diverse. Encapsulins are made of one type of shell protein, as opposed to several as is observed in many bacterial microcompartments (8, 9). The key feature of encapsulin systems is that cargo proteins can be specifically encapsulated and targeted to the encapsulin capsid interior, using a selective C-terminal sequence referred to as targeting peptides (TPs) (10). The functions of the nanocompartment are associated with the functions of its protein cargo. Many functionally diverse cargo proteins are associated with encapsulins, including dye-decolorizing peroxidases (DyPs) (11), ferritin-like proteins (FLP) (12), hydroxylamine oxidoreductase (HAO) (13), and cysteine desulfurases (14). Moreover, it has been shown that some encapsulin systems may possess multiple cargo proteins, which are made up of one core cargo protein and up to three secondary cargo proteins according to the TPs (6). Notably, a large proportion of native cargo proteins are DyP-type peroxidases, conferring the resistance of the cell to oxidative stress (6, 7, 11, 15–18). However, to date, the structural information on the cargo-encapsulated encapsulins is not yet available (SI Appendix, Table S1), and thus, little is known about the structural arrangement and mechanistic features of the cargo proteins loaded in the encapsulins.Actinobacteria harbors the largest number of encapsulin or encapsulin-like systems (6). DyP-containing encapsulins have already been reported from mycobacteria, including Mycobacterium smegmatis (15) and Mycobacterium tuberculosis (19). These have been considered as potential biomarkers to detect active tuberculosis (TB) (20). In the present study, we have isolated and characterized a DyP-loaded encapsulin system from M. smegmatis, which is commonly used as a model organism in studying the biology of the M. tuberculosis (21). We have determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). Our results have revealed the interactions between the CFP-29 (a 29 kDa culture filtrate protein) shell and DyP cargo and a potential antioxidation mechanism. Our study also lays the foundation for the discovery of new diagnosis protocols and treatments of TB.     

间日疟原虫pvMSP_(1-19)基因重组耻垢分枝杆菌疫苗的构建与鉴定

目的体外扩增间日疟原虫pvMSP1-19基因,构建大肠埃希菌-穿梭质粒PMV261/pvMSP1-19和电转化耻垢分枝杆菌。方法采用聚合酶链反应(PCR),体外扩增间日疟原虫的pvMSP1-19基因,克隆入PMD19-T载体,构建亚克隆PMV/pvMSP1-19大肠埃希菌-分枝杆菌穿梭质粒;电转化方法将PMV/pvMSP1-19穿梭质粒转化到耻垢分枝杆菌中。并热诱导此重组分枝杆菌,SDS-PAGE电泳观察pvMSP1-19蛋白的表达,Western blot鉴定其免疫学活性。结果成功扩增了间日疟原虫pvMSP1-19基因,并且正确构建PMV/pvMSP1-19穿梭质粒和转化耻垢分枝杆菌。采用Westernblot证实该重组耻垢分枝杆菌表达的pvMSP1-19蛋白能被间日疟患者的血清识别。结论构建的重组耻垢分枝杆菌能表达pvMSP1-19蛋白,该蛋白具有免疫反应性,为pvMSP1-19基因重组BCG疫苗的研制提供了必要的基础。     

结核分枝杆菌肝素结合血凝素与人IL12融合基因重组耻垢分枝杆菌的构建及鉴定

目的构建表达肝素结合血凝素与人IL12融合基因的重组耻垢分枝杆菌并对其进行鉴定。方法通过RT-PCR从人淋巴细胞获得hIL12基因模板,采用PCR技术克隆hIL12的P40和P35两个亚基及结核分枝杆菌的HBHA基因,通过酶切鉴定及DNA测序证实连接的基因片段均正确后,构建分枝杆菌穿梭表达质粒pSMT3-HBHA-hIL12,并用电穿孔法将重组质粒转到耻垢分枝杆菌中。分别用HBHA单克隆抗体和hIL12多克隆抗体检测HBHA和hIL12蛋白在重组耻垢分枝杆菌中的表达,并测定重组耻垢分枝杆菌生长状态的改变。结果重组耻垢分枝杆菌构建成功,通过HBHA单抗和hIL12多抗进行免疫荧光法检测证实HBHA和hIL12均有表达,并且重组的耻垢分枝杆菌的生长速度比正常组有较大的提高。结论成功构建了表达结核分枝杆菌HBHA基因和人IL12的重组耻垢分枝杆菌,为构建一种结核病的候选疫苗提供了实验基础。     

重组卡介苗的研究与应用

重组卡介苗可同时表达多种病原体抗原，已成为新的疫苗载体。对重组卡介苗的研究方法和进展，包括重组卡介苗疫苗载体、启动子、电转化方法、绿色荧光蛋白的作用、重组耻垢分枝杆菌疫苗及重组卡介苗的作用机制等进行综述，并介绍了重组卡介苗在表达病毒、细菌及寄生虫抗原和表达细胞因子及抗肿瘤等方面的应用。     

Synthesis of curcumin-loaded chitosan phosphate nanoparticle and study of its cytotoxicity and antimicrobial activity

Curcumin has acquired an important position in the treatment of various diseases. But its use, as a chemotherapeutic agent, is limited due to its low water solubility, poor bioavailability, and its sensitive nature at the physiological pH. To overcome this, curcumin was loaded into chitosan phosphate nanoparticles (CPNs). The loading efficiency was found to be 84%. DLS studies revealed the average particle size of CPNs and curcumin-loaded CPNs as 53 and 91 nm, respectively, and TEM results supplemented these values. A sustained release pattern was noticed and the amount of curcumin released in acidic pH was higher than at physiological pH. The curcumin nanoformulation exhibited proficient activity against both Gram-positive and Gram-negative bacteria as well as fungus. Cytocompatibility of the nanoformulations against peripheral blood mononuclear cells (PBMCs) and murine monocyte–macrophage cell line was confirmed by incubating with PBMCs and murine monocyte–macrophage cell line.     

人类乳头瘤病毒16型E7C亚基因在耻垢分枝杆菌中的表达

目的在耻垢分枝杆菌Mycobaterium smegmatis mc2155中表达人类乳头瘤病毒16型E7C亚基因,为其重组BCG疫苗的研究奠定基础。方法采用电穿孔转化法将重组质粒pBCG-E7C导入耻垢分枝杆菌(M.smegmatismc2155)中,通过卡那霉素抗性筛选经PCR鉴定的重组M.smegmatis mc2155,培养于middlebrook7H9 broth(M7H9)培养基中,并添加10%M7H9 enrichment ADC和0.05%Tween 80,37℃培养48 h~72 h,42℃诱导表达5 h,表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析。结果成功构建pBCG3000-E7C质粒,SDS-PAGE显示表达产物的分子质量单位约为6.5 ku,与预期理论值相符,Western blot分析表达产物能被宫颈癌患者血清识别。结论人类乳头瘤病毒16型E7C基因在M.smegmatis mc2155中成功表达。     

耻垢分枝杆菌中DNA损伤与SOS反应的实验研究

目的探讨基因毒性物质介导的DNA损伤引起分枝杆菌SOS反应的作用。方法选择耻垢分枝杆菌为分枝杆菌模式细菌,通过紫外线损伤试验检测耻垢分枝杆菌对紫外线的敏感性,通过2倍稀释法测定利福平和氧氟沙星的最小抑菌浓度（MIC）。分别用紫外线和次抑菌浓度（1/2MIC）抗生素处理耻垢分枝杆菌,在不同时间点收集菌液,以看家基因sigA为内参,采用实时定量PCR相对定量方法检测dnaE2、recA和recX等SOS基因表达水平的变化。结果紫外线暴露45s后耻垢分枝杆菌的存活率为50%,利福平和氧氟沙星的MIC分别是32μg/ml和1μg/ml。耻垢分枝杆菌dnaE2和recX等SOS基因表达水平在紫外线下暴露45s后即开始上升,recA的表达水平在紫外线暴露3h后显著上升。耻垢分枝杆菌dnaE2和recX基因表达水平在1/2MIC的利福平作用后1h或在1/2MIC的氧氟沙星作用0h后上升,recA的表达水平在1/2MIC的利福平或氧氟沙星作用3h时显著升高。结论紫外线、抗生素等基因毒性物质可以导致耻垢分枝杆菌DNA损伤及SOS基因表达水平上升。     

Mycobacterium smegmatis pneumonia

Mycobacterium smegmatis is a non-tuberculous mycobacterium that is usually associated with soft tissue or wound infections in humans. Pulmonary infections secondary to this pathogen are rarely seen and occur only in patients with an underlying condition, such as lipoid pneumonia. This report presents the first case of M. smegmatis pneumonia in an otherwise healthy individual who had no predisposing condition.