Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus

Background:CE is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods:The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results:Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion:Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice. Key Words: Echinococcus granulosus, Lactococcus lactis, Immunization, Vaccines     

褐飞虱小分子量热激蛋白基因表达特性和功能

【目的】研究褐飞虱小分子量热激蛋白的表达特性和功能，明确其在褐飞虱温度胁迫适应中的作用。【方法】采用BLAST从转录组数据库中筛选褐飞虱小分子量热激蛋白基因序列；利用Bioedit、Mega等分子生物学软件进行序列分析；利用qPCR技术分析目的基因在不同处理下的表达特性；利用原核表达技术研究其功能。【结果】筛选到6个含有α-晶体结构的小分子量热激蛋白基因NlHsp20.9、NlHsp21.6、NlHsp21.9、NlHsp22.4、NlHsp23.1、NlHsp28.7，其ORF长度依次为561、531、570、570、588和735 bp，理论等电点为5.96、5.77、6.32、5.01、5.74和7.74。NlHsp28.7在3龄若虫中的表达量最高，而NlHsp21.9和NlHsp23.1在雌成虫中的表达量最高。雌虫在低温胁迫后所有小分量热激蛋白基因的表达量均下降，高温胁迫后除NlHsp22.4外的其他5个基因表达量不同程度上调；3龄若虫在低温胁迫后一半sHsps表达量下降，另一半上升，高温胁迫后全部上调。转化褐飞虱sHsps的重组大肠杆菌热激存活率显著上升。【结论】褐飞虱小分子量热激蛋白具有龄期和诱导表达特性及热胁迫保护功能，可能在其高温胁迫应激中具有重要作用，在低温胁迫应激中的作用与虫态有关。     

褐飞虱小分子量热激蛋白基因表达特性和功能

【目的】研究褐飞虱小分子量热激蛋白的表达特性和功能,明确其在褐飞虱温度胁迫适应中的作用。【方法】采用BLAST从转录组数据库中筛选褐飞虱小分子量热激蛋白基因序列;利用Bioedit、Mega等分子生物学软件进行序列分析;利用qPCR技术分析目的基因在不同处理下的表达特性;利用原核表达技术研究其功能。【结果】筛选到6个含有α-晶体结构的小分子量热激蛋白基因NlHsp20.9、NlHsp21.6、NlHsp21.9、NlHsp22.4、NlHsp23.1、NlHsp28.7,其ORF长度依次为561、531、570、570、588和735 bp,理论等电点为5.96、5.77、6.32、5.01、5.74和7.74。NlHsp28.7在3龄若虫中的表达量最高,而NlHsp21.9和NlHsp23.1在雌成虫中的表达量最高。雌虫在低温胁迫后所有小分量热激蛋白基因的表达量均下降,高温胁迫后除NlHsp22.4外的其他5个基因表达量不同程度上调;3龄若虫在低温胁迫后一半sHsps表达量下降,另一半上升,高温胁迫后全部上调。转化褐飞虱sHsps的重组大肠杆菌热激存活率显著上升。【结论】褐飞虱小分子量热激蛋白具有龄期和诱导表达特性及热胁迫保护功能,可能在其高温胁迫应激中具有重要作用,在低温胁迫应激中的作用与虫态有关。     

小麦热激蛋白转录因子C家族基因的结构及其在干旱胁迫中的表达

小麦热激蛋白转录因子在热胁迫和耐热性产生的过程中发挥重要作用。为进一步了解小麦热激蛋白转录因子C亚家族,本研究采用全基因组信息保守结构域比对和进化聚类分析,共鉴定了24个小麦TaHsfC基因,并对TaHsfC亚家族成员染色体定位、基因结构、亚细胞定位、对应蛋白质的氨基酸特点、基因表达进行了分析。结果表明,24个TaHsfCs主要分布在5条染色体上,其中A基因组中有7个,B基因组中9个,D基因组中有6个,另外两个所在染色体位置不清楚。TaHsfCs含有0～2个内含子,其中 TaHsfC1亚族基因含有1～2个内含子, TaHsfC2亚族基因则含有0～1个内含子。聚类分析表明,小麦TaHsfC成员共分为 TaHsfC1和 TaHsfC2两个亚族。24个TaHsfCs成员全部定位于细胞核。在15%PEG模拟干旱条件下,干旱敏感品种矮抗58和耐旱品种晋麦47中有10个TaHsfC基因显著上调表达,其中 TaHsfC2亚族基因的表达量上调倍数均高于 TaHsfC1亚族基因(其中 TaHsfC2j和 TaHsfC2k未在晋麦47中检测到表达量)。本研究可为探索小麦热激蛋白转录因子C家族基因在小麦抗旱中的分子机制提供一定的理论支撑。     

热激对玉米大斑病菌生长和发育的影响

通过对玉米大斑病菌进行热激处理,观测热激对病菌孢子萌发、菌丝生长速度、菌落形态、菌丝形态等方面的影响。结果表明:热激处理对分生孢子萌发率的影响与热激温度有关,42℃热激促进孢子萌发,45℃热激对孢子萌发没有显著影响,48℃热激则会显著抑制孢子萌发;42℃～48℃热激处理会降低病菌的菌落生长速度,热激温度越高菌落生长速度越慢;42℃～48℃热激对菌丝形态、分生孢子形态、菌落颜色和菌落形态均无明显影响,但菌丝细胞中的内容物减少,有时出现内容物缺失的菌丝细胞。     

小麦冷休克蛋白基因TaCSP的克隆及表达分析

为深入研究小麦冷休克蛋白(cold shock proteins,CSPs)基因,根据大肠杆菌(E.coli)CSPs蛋白保守氨基酸序列,借助生物信息学技术检索小麦EST序列,采用同源序列法拼接了小麦3个冷休克蛋白基因,并以小麦叶片总RNA为模板,采用RT-PCR对其进行扩增;同时,采用荧光定量PCR对3个基因的组织表达特性以及ABA、低温、高温、干旱和盐胁迫条件下的表达模式进行了研究。结果表明,小麦3个冷休克蛋白基因TaCSP1、TaCSP2和TaCSP3全长分别为290、374和377bp,各编码69、69、80个氨基酸残基。序列分析表明,TaCSP1、TaCSP2和TaCSP3之间氨基酸相似性为72.9%~84.3%,与大肠杆菌来源CSPs的相似性为40.0%~79.7%。荧光定量PCR表达谱分析显示,TaCSPs在抽穗期小麦的根、茎、叶和幼穗中均能表达。胁迫分析表明,3个冷休克蛋白基因在小麦幼根中的表达,在低温和ABA处理下,表现为早期诱导、后期抑制,在高温、干旱和盐胁迫下,则表现为早期抑制、后期有所恢复。克隆获得的小麦3个冷休克蛋白基因在根组织中强烈表达,并受ABA和冷胁迫诱导,推测其在小麦抵御逆境胁迫过程中发挥重要作用。     

Influence of Nigella sativa fixed oil on some blood parameters and histopathology of skin in staphylococcal-infected BALB/c mice

Nigella sativa has been used for a long time in Jordanian folk medicine to treat skin diseases like microbial infections and inflammation. Therefore, the present study was conducted to assess the healing efficacy of petroleum ether extract of Nigella sativa seeds (fixed oil) on staphylococcal-infected skin. Male BALB/c mice were infected with 100 microL of Staphylococcus aureus (ATCC 6538) suspension at a dose of 10(8) colony forming unit/mouse into shaved mild dorsal skin. Application of treatments for each group (100 microL sterile saline, 100 microL chloramphenicol (10 microg/mouse) and Nigella sativa fixed oil at a dose of 50, 100 or 150 microL/mouse) was performed at the site of infection twice a day for two consecutive days after 3 h of infection. At day 3 and 5 after infection, total White Blood Cells (WBCs) count; differential and absolute differential WBC counts and the number of viable bacteria present in the skin area were measured. At day 5 after infection, the animals were sacrificed and the histology of skin was examined. Results indicated that fixed oil of Nigella sativa seeds enhance healing of staphylococcal-infected skin by reducing total and absolute differential WBC counts, local infection and inflammation, bacterial expansion and tissue impairment. These effects provide scientific basis for the use of Nigella sativa in traditional medicine to treat skin infections and inflammations.     

玉米热激蛋白ZmHSP70-12基因的克隆及表达分析

热激蛋白70(HSP70)是原核和真核细胞中普遍存在的一种高度保守的分子伴侣。从玉米中克隆1个HSP70家族成员。该基因cDNA序列全长为1 992 bp,开放阅读框为2 352 bp,编码663个氨基酸,蛋白质分子量约75.0 kD。蛋白结构预测及同源比对分析表明,该基因编码蛋白含ATPase位点和HSP70保守结构域,与拟南芥AtHSP70-12序列高度相似,命名为ZmHSP70-12。蛋白亚细胞定位显示,ZmHSP70-12蛋白在内质网中表达。实时荧光定量PCR分析表明,ZmHSP70-12对非生物胁迫高温、干旱均具有明显的应答反应,推测ZmHSP70-12是玉米中与胁迫逆境相关的基因。     

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Background: Herpes simplex virus type-1 (HSV-1) establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3''-diaminobenzidine (DAB) substrate. Methods: Eight-week-old male BALB/c mice were inoculated via the eye by 10⁴ plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. Results and Conclusion: The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia. Key Words: Herpes simplex virus-1, Latency, In situ PCR, two-round PCR, Trigeminal ganglia     

Inhibitory effect of whole oat on aberrant crypt foci formation and colon tumor growth in ICR and BALB/c mice

Recently, the incidence of colon cancer has been rapidly increasing in previously low-risk countries other than the Western world. Since dietary factors are thought to be key components involved in high risk colon cancer, the current trend for colon cancer prevention is toward dietary intervention. To explore if whole oat functions as a chemoprevention agent, an inflammation-related mouse colon cancer model, initiated with 1, 2-dimethylhydrazine (DMH), followed by dextran sodium sulfate (DSS), was performed to evaluate the preventive effect of whole oat containing diets. The result indicated middle and high dose whole oat diets significantly reduced the number of aberrant crypt foci (ACF) as well as colon tumors. Further, human colon carcinoma cells were subcutaneously inoculated into BALB/cAnNg-Foxn1 nude mice to measure the growth inhibition on whole oat diets. Low, middle and high dose whole oat diets significantly decreased the tumor volumes by 13%, 17% and 43%, respectively, indicating a dose dependent inhibitory effect. Meanwhile, 38% and 54% reductions in tumor weights were observed in middle and high dose whole oat diets. Together, the evidence suggests whole oat helps protect against colon cancer development and could be a good chemoprevention agent taken as a daily supplement.     

人参地黄桑葚片对小鼠免疫功能的影响

目的探索人参地黄桑葚片对小鼠免疫功能的影响。方法将雄性ICR小鼠随机分为对照组、人参地黄桑葚片高剂量组(2.250g/kg)、人参地黄桑葚片中剂量组(0.750g/kg)、人参地黄桑葚片低剂量组(0.375g/kg),每组15只,连续灌胃人参地黄桑葚片30天,称量各组小鼠免疫重量并计算器官指数、测定绵羊红细胞(SRBC)诱导的迟发型变态反应(DTH)、自然杀伤(NK)细胞的活性、脾淋巴细胞增殖能力、腹腔巨噬细胞对鸡红细胞(CRBC)的吞噬能力。结果人参地黄桑葚片高、中、低剂量组均能显著增加小鼠胸腺指数及脾脏指数(P0.05);人参地黄桑葚片高、中剂量组能显著增强小鼠NK细胞活性及SRBC诱导的小鼠DTH(P0.05);人参地黄桑葚片高剂量组能显著提高腹腔巨噬细胞对CRBC的吞噬作用及Con A诱导的脾淋巴细胞的增殖力(P0.05)。结论人参地黄桑葚片对小鼠的免疫器官指数、NK细胞活性、腹腔巨噬细胞对CRBC的吞噬作用、Con A诱导脾淋巴细胞的增殖能力以及SRBC诱导的小鼠DTH具有的提高作用。     

植物热激转录因子及其与耐热性关系的研究进展

热激转录因子是高等植物热胁迫响应基因转录水平上的中心调控蛋白,在植物的热胁迫信号转导以及耐热性中起着关键作用。为了给作物耐热性研究和应用提供有益的参考,本文从热激转录因子家族基因的分类、克隆、功能分析及其作用机理等方面详细介绍了模式植物拟南芥以及番茄、水稻、玉米和小麦等主要作物中该家族基因与耐热性关系的研究现状,并对该家族基因研究中亟待解决的问题进行了探讨。     

高温胁迫下茄子耐热性表现及耐热指标的筛选

对7份茄子材料分别进行室内高温鉴定和田间自然高温鉴定,并探讨2种鉴定方式的相关性,以筛选适宜、快速、准确的鉴定指标。结果表明：除可溶性糖外,幼苗热害指数、细胞膜相对电导率、脯氨酸含量、丙二醛（MDA）可有效的评价不同茄子幼苗的耐热性。田间高温条件下,经室内鉴定为耐热品种的正常花率和坐果率均高于不耐热品种。经相关分析结果可知,各品种的正常花率和坐果率与苗期热害指数、细胞膜相对电导率、脯氨酸含量、MDA含量呈显著性相关。本研究结果表明细胞膜相对电导率、脯氨酸含量、MDA及热害指数可作为茄子耐热性快速鉴定的指标。     

翘鳞伞胞外多糖的抗肿瘤活性和对荷瘤小鼠免疫功能的影响

为考察翘鳞伞胞外多糖对荷瘤小鼠体内肿瘤的抑制及其免疫功能的影响;采用无机陶瓷膜从翘鳞伞(Pholiota squarrosa)AS 5.245液体发酵醪中分离得到的胞外粗多糖(Crude Extracellular Polysaccharide,CEP),以荷瘤S180小鼠为试验对象,用免疫器官重量法,进行高、中、低剂量CEP的腹腔注射试验;试验结果表明,CEP具有较高的抑瘤活性,并呈剂量相关性;中、高剂量组与对照组相比较,小鼠胸腺指数和脾脏指数均有显著增加(P0.05);与低剂量组相比较,亦均有显著增加;表明CEP可以增强荷瘤小鼠的免疫功能对肿瘤进行抑制。     

茯砖茶对小鼠肠道免疫功能调节作用的研究

探讨了茯砖茶水提物对小鼠肠道免疫功能的调整作用。试验以注射用氨苄青霉素灌胃建立小鼠肠道菌群失调及免疫功能紊乱模型。昆明小鼠随机分6组,正常对照组、自然恢复组、模型组和茯砖茶高、中、低剂量组,分别进行肠道菌群检测、小肠黏液sIgA的测定、血清中IL-2的测定、血清白蛋白和总蛋白含量检测以及回肠切片病理检测。结果表明,注射用氨苄青霉素灌胃5 d小鼠,肠道菌群失调,小肠黏液sIgA、血清中IL-2、血清白蛋白和总蛋白均较正常组降低,回肠组织切片明显病变。分别灌服茯砖茶水提物后,肠道菌群得以调整,小肠黏液sIgA、血清中IL-2、血清白蛋白和总蛋白均有所升高,且与自然恢复组比具有显著性差异。各治疗组之间比较,高剂量组疗效优于低剂量组。说明茯砖茶水提物能修复受损黏膜,有调节肠道免疫功能的作用。     

Immune Modulating Brevetoxins: Monocyte Cytotoxicity,Apoptosis, and Activation of M1/M2 Response Elements Is Dependent on Reactive Groups

Brevetoxins are a suite of marine neurotoxins that activate voltage-gated sodium channels (VGSCs) in cell membranes, with toxicity occurring from persistent activation of the channel at high doses. Lower doses, in contrast, have been shown to elicit neuroregeneration. Brevetoxins have thus been proposed as a novel treatment for patients after stroke, when neuron regrowth and repair is critical to recovery. However, findings from environmental exposures indicate that brevetoxins may cause inflammation, thus, there is concern for brevetoxins as a stroke therapy given the potential for neuroinflammation. In this study, we examined the inflammatory properties of several brevetoxin analogs, including those that do and do not bind strongly to VGSCs, as binding has classically indicated toxicity. We found that several analogs are toxic to monocytes, while others are not, and the degree of toxicity is not directly related to VGSC binding. Rather, results indicate that brevetoxins containing aldehyde groups were more likely to cause immunotoxicity, regardless of binding affinity to the VGSC. Our results demonstrate that different brevetoxin family members can elicit a spectrum of apoptosis and necrosis by multiple possible mechanisms of action in monocytes. As such, care should be taken in treating “brevetoxins” as a uniform group, particularly in stroke therapy research.     

Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy

Background:

Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, ¹H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major.

Methods:

Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. ¹H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis.

Results:

The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid.

Conclusion:

¹H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. Key Words: Leishmania major, Metabolomics, Principal component analysis     

小麦TCP基因家族的全基因组鉴定和对热胁迫的响应

TCP是植物发育调控的重要转录因子。为了挖掘小麦TCP基因的功能,本研究利用生物信息学方法和qRT-PCR技术,对小麦TCP基因家族进行全基因组鉴定和在热胁迫下的表达分析。全基因组鉴定结果表明,普通小麦品种中国春中存在58个非冗余的TaTCP基因。根据与AtTCPs和OsTCPs的系统发育分析,在小麦中分别有26个PCF、20个CIN和12个CYC/TB1聚类。进一步预测TaTCP基因在达尔文进化下的压力选择,发现除了TaTCP16-A相对于乌拉尔图小麦中直系同源基因是正向选择,其他基因都是纯化选择的过程。利用在线RNA-seq数据分析TaTCPs基因在5种组织器官中与冷、热和干旱胁迫下的表达图谱,结果显示,小麦TCP基因在不同的组织和胁迫中存在差异表达。最后,通过qRT-PCR技术检测12个不同发育阶段的小麦叶片在短期热胁迫下TaTCP5部分同源基因的表达水平,结果表明,在35℃下处理1h,TaTCP5-D的相对表达量在拔节期、拔节后期、孕穗前期和抽穗期都有明显的上调,这些结果为进一步研究TaTCP基因的功能奠定了良好的基础。