Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin(OVA)is the major allergenic protein that can induce T helper 2(Th2)-allergic reactions,for which current treatment options are inadequate.In this study,we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid(Lac/CA)-catalyzed crosslinking in conjunction with galactomannan(Man).The formation of high molecular weight crosslinked polymers and the Ig G-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity.Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF).Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines(interleukin(IL)-4,IL-5,and IL-13),and upregulated IFN-γand IL-10 expression.Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers(CD80 and CD86)and MHC class II molecules,and suppressed the expression levels of proinflammatory cytokines.The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Investigation into IgG/IgE binding capacity and gut microbiota of digestion products derived from glycated ovalbumin

Gut microbiota plays an important role in food allergy.The immunoglobulin G(IgG)/immunoglobulin E(IgE)binding capacity and human gut microbiota changes of digestion products derived from glycated ovalbumin(OVA)were investigated.Gastrointestinal digestion effectively destroyed the primary structure of glycated OVA,resulting in a significantly higher digestibility than gastric digestion,and more abundant peptides<3 kDa.Moreover,gastric and gastrointestinal digestion products have different fluorescence quenching and red shift of fluorescence peaks,and possess different conformational structures.These changes resulted in a decrease in 28.7%of the IgE binding capacity of gastrointestinal digestion products beyond that of pepsin.Moreover,gastrointestinal digestion products of glycated OVA increased significantly the proportion of Subdoligranulum,Collinsella,and Bifidobacterium.Therefore,gastrointestinal digestion products of glycated OVA altered human intestinal microbiota,reducing the risk of potential allergy.

Rosmarinic acid improves tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats

Objective:To evaluate the effect of rosmarinic acid on tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats.Methods:Rats were randomly divided into six groups:the control group,the asthmatic group,and the asthmatic groups treated with dexamethasone(1 mg/kg;oral gavage)or three doses of rosmarinic acid(0.5,1,and 2 mg/kg;oral gavage).For induction of asthma,rats received intraperitoneal injections and inhalation of ovalbumin.After 21 days,bronchoalveolar lavage fluid and lung samples were collected for histopathological analyses.Moreover,total and differential white blood cell counts were determined.Results:The rosmarinic acid-treated group had significantly lower tracheal smooth muscle responses to methacholine than the asthmatic group.In addition,rosmarinic acid reduced white blood cell count and the percentages of eosinophils,monocytes,and neutrophils while increasing the percentage of lymphocytes.Ovalbumin-induced lung pathological changes were significantly improved by treatment with rosmarinic acid.Conclusions:Rosmarinic acid improves tracheal smooth muscle responsiveness and lung pathological changes in ovalbumin-sensitized rats.

基于酵母β-葡聚糖载体的口服OVA疫苗(WGP-OVA疫苗)诱导体液免疫及抗原特异性T细胞免疫反应

目的:探讨口服WGP-OVA疫苗对C57BL/6小鼠体液免疫及抗原特异性T细胞免疫的诱导作用。方法:分别连续给予C57BL/6小鼠口服PBS、WGP及WGP-OVA 17 d后,使用LEGENDplexTM多因子流式检测试剂盒检测给药后小鼠血清中IgG1、IgG2a、IgG3及IgM的含量;HE染色比较各组小鼠脾脏淋巴小结的大小。取小鼠腹股沟淋巴结(ILNs)制备单细胞悬液,流式细胞仪(FACS)检测淋巴结内F4/80+巨噬细胞及F4/80+SIINFEKL+巨噬细胞比例,分析WGP-OVA对巨噬细胞在ILNs内的浸润及抗原提呈情况;同时通过MHC Tetramer染色检测抗原特异性CD8+T细胞比例,明确WGP-OVA疫苗对T细胞免疫的诱导作用。结果:与PBS组相比,WGP-OVA能显著诱导IgG1、IgG2a、IgG3及IgM的分泌,增大脾脏内淋巴小结。此外,口服WGPOVA疫苗能促进F4/80+巨噬细胞向淋巴结浸润,诱导巨噬细胞对OVA的抗原提呈,同时增加淋巴结内OVA特异性CD8+CTLs的数量。结论:口服WGP-OVA疫苗能诱导C57BL/6小鼠体液免疫及抗原特异性T细胞免疫反应。

Myricetin alleviates ovalbumin-induced allergic rhinitis in mice by regulating Th1/Th2 balance

Objective:To evaluate the effect of myricetin on ovalbumin(OVA)-induced allergic rhinitis in mice.Methods:Mice were sensitized and challenged using OVA(5%,500 mL)intraperitoneally and intranasally,respectively,on an alternative day for 14 days,followed by administration of myricetin(50,100,and 200 mg/kg)till day 21.Nasal symptoms,biochemical parameters,protein expressions,and histopathology were observed.Results:OVA-induced increased nasal symptoms including rubbing,sneezing,and discharge were significantly reduced by myricetin(100and 200 mg/kg)(P<0.05).Myricetin also protected against histamine challenge and attenuated elevated serum immunoglobulin E(IgE;total and OVA-specific),total IgG1,andβ-hexosaminidase levels,as well as leukotriene C4 and interleukins levels in nasal lavage fluid(P<0.05).Western blot analysis showed that myricetin significantly upregulated the protein expression of T-box expressed in T cells,while downregulating the protein expression of GATA binding protein 3,NF-κB,and IκB-α(P<0.05).Additionally,OVA-induced histopathological abberations in the nasal mucosa was markedly ameliorated by myricetin treatment(P<0.05).Conclusions:Myricetin exerts anti-allergic effects against OVAinduced allergic rhinitis via regulating Th1/Th2 balance.

自组装纳米颗粒肿瘤疫苗OVA257-264-mi3的构建及其保护效果评价

目的构建SpyCatcher-mi3纳米颗粒疫苗递送载体,评价其在增强卵清蛋白CD8+T细胞表位肽OVA 257-264免疫原性和在小鼠荷瘤模型中免疫保护效果。方法SpyCatcher-mi3蛋白由大肠杆菌表达,依次经亲和层析、阴离子交换层析纯化得到;合成OVA 257-264-SpyTag多肽,通过SpyTag/SpyCatcher蛋白质连接系统构建纳米颗粒OVA 257-264-mi3;通过细胞溶血实验和细胞毒性实验评价OVA 257-264-mi3体外安全性,记录小鼠体重变化和镜检脏器组织切片HE染色评价OVA 257-264-mi3体内安全性;将6~8周龄,18~20 g,SPF级,雌性C57BL/6小鼠按随机数字表法分为OVA 257-264-mi3组、OVA 257-264组和Control组,每组14只,分别于第0、14、28天免疫小鼠,末次免疫后第14天通过ELISpot检测其脾脏淋巴细胞中IFN-γ分泌细胞数量评价其免疫原性;在小鼠荷瘤模型上通过核磁共振成像等手段评价纳米颗粒疫苗OVA 257-264-mi3的保护效果。结果SpyCatcher-mi3和OVA 257-264-mi3均自组装形成均一稳定的纳米颗粒,平均粒径约为43.8 nm和91.3 nm;OVA 257-264-mi3在体内外均具有良好的安全性;OVA 257-264-mi3组小鼠每1×106个脾脏淋巴细胞中IFN-γ分泌细胞数量达253,显著高于OVA 257-264组(P<0.05);OVA 257-264-mi3组小鼠第22天荷瘤体积约151.1 mm 3,显著小于OVA 257-264组(P<0.05),且观察期内生存率达60%,显著高于OVA 257-264组(P<0.05)。结论成功构建了纳米颗粒疫苗OVA 257-264-mi3,其增强肿瘤抗原表位肽OVA 257-264免疫原性,在小鼠荷瘤模型中保护效果良好,为肿瘤新抗原疫苗的研究提供了理论基础。

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

太子参多糖对OVA免疫小鼠肠道免疫应答能力的影响

【目的】研究太子参多糖(PHPS)对卵清白蛋白(OVA)免疫小鼠肠道免疫应答的影响,为了解PHPS能否增强小鼠机体的免疫应答能力提供依据。【方法】将72只健康清洁级(SPF)雌性ICR小鼠随机分成空白对照组(CK组)、OVA免疫对照组(OVA组)、铝佐剂阳性对照组(AL组)及PHPS各剂量组[50 mg·kg^(-1) PHPS(PHPS-L)组、100 mg·kg^(-1) PHPS(PHPS-M)组、200 mg·kg^(-1) PHPS(PHPS-H)组],每组12只小鼠。试验第1~5天和第16~20天,PHPS各剂量组小鼠灌胃对应剂量的PHPS,其余各组均灌胃双蒸水;试验第6天和第21天,CK组小鼠按0.2 mL·只^(-1)的剂量腹股沟皮下注射无菌PBS,AL组小鼠按0.2 mL·只^(-1)的剂量腹股沟皮下注射OVA与铝佐剂的混合液,其他组小鼠均按0.2 mL·只^(-1)的剂量腹股沟皮下注射OVA溶液;试验第36天采用颈椎脱臼法处死小鼠,采集样品,制作十二指肠、空肠、回肠切片检测小鼠肠道形态结构和上皮内淋巴细胞数量,检测小鼠肠道细胞因子(IL-6、TNF-α)和分泌性免疫球蛋白A(SIgA)的含量,以及IL-6、TNF-αmRNA的转录水平。【结果】(1)与OVA组相比,PHPS各剂量组小鼠十二指肠、空肠、回肠的绒毛高度和绒隐比提高,PHPS-M组十二指肠、PHPS-H组空肠、PHPS各剂量组回肠的隐窝深度显著降低(P<0.05);同时,PHPS各剂量组十二指肠、空肠、回肠的上皮内淋巴细胞数量均有所增加。(2)与OVA组相比,PHPS各剂量组小鼠十二指肠、空肠、回肠IL-6、TNF-α的含量增加,SIgA含量显著增加(P<0.05)。(3)与OVA组相比,PHPS各剂量组小鼠十二指肠、空肠、回肠IL-6、TNF-αmRNA的转录水平显著上调(P<0.05)。【结论】PHPS能在一定程度上改善肠道形态结构,促进肠道上皮内淋巴细胞分泌,调节相关免疫分子的释放与表达,从而增强肠道黏膜免疫反应,提高小鼠对OVA的免疫应答能力。

Oral administration of Bacillus coagulans TQ-35 alleviates allergic responses in OVA-sensitive BALB/c mice

Bacillus coagulans has been extensively studied so far,but there has been a lack of research on its usage in allergy.In this study,we designed to assess the effect of different concentrations of B.coagulans on food allergy in a BALB/c mouse model of ovalbumin(OVA)-induced food allergy and its effect on gut microbes.The assessment of symptoms,specific immunoglobulin E(IgE),T-cell differentiation,and related gene expression levels in sensitized mice by assay indicated that high doses of oral B.coagulans could alleviate allergic symptoms.Treatment with B.coagulans,in the high-dose group,significantly reduced IgE and IgG1 levels and modulated the balance of T helper type 1 cell(Th1)and Th2 and the expression of relevant genes in the spleen.16S rRNA analysis showed that probiotics improved the structure of the microbiota,in particular by boosting the percentage of Clostridia,Bacteroides vulgatus and Enterococcus faecium,and by increasing the abundance of microbial species,thereby modulating the immune system.Therefore,this study can provide insights into the practical application of B.coagulans doses to alleviate OVA allergy.

芒柄花素磺酸钠通过MAPKS信号通路缓解OVA诱导哮喘小鼠肺部炎症

[目的]观察芒柄花素磺酸钠对卵清蛋白(OVA)诱导哮喘小鼠的保护作用及其机制。[方法]购买SPF级C57BL/6小鼠60只,并随机分6组,每组10只,具体组别如下:空白对照组(CON组)、模型组(OVA组)、芒柄花素磺酸钠低剂量(MBHS-L组)、芒柄花素磺酸钠中剂量(MBHS-M组)、芒柄花素磺酸钠高剂量(MBHS-H组)和地塞米松组(DEX组),使用OVA诱导建立小鼠哮喘模型。实验主要评价小鼠哮喘症状并进行评分、计量支气管肺泡灌洗液(BALF)中炎症细胞数量(Diff-Quick染色法)、镜下观察肺组织的病理学变化[苏木精-伊红(HE)染色法]、分析BALF中超氧化物歧化酶(SOD)活性和肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和丙二醛(MDA)水平(ELISA法和化学发光法)、分析肺组织p-ERK1/2,p-JNK和p-p38MAPK蛋白表达水平(免疫荧光法和Western blot法)。[结果]与OVA组比较,哮喘症状评分、BALF炎症细胞数量、TNF-α、IL-1β和MDA水平以及p-ERK1/2,p-JNK和p-p38MAPK蛋白表达水平在MBHS组和DEX组均明显降低(P<0.05),而SOD的活性明显升高(P<0.05),肺组织病理学变化明显改善。[结论]芒柄花素磺酸钠可能通过MAPKS信号通路缓解OVA诱导哮喘小鼠的肺部炎症。

Preliminary Study on the Separation of Lysozyme,Ovotransferrin and Ovalbumin from Egg White

[Objective] A study on separation process of lysozyme, ovotransferrin and ovalbumin from egg white. [Method] The proteins were separated by ammonium sul-fates and ion-exchange chromatography. Purity of the proteins was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. [Result] The results showed that the proteins were electrophoresis-pure. The specific activity of lysozyme was increased from 144.13 to 2 235 U/mg, and purification factor was 15-fold. Lysozyme recovery rate was estimated to be 15.76%. Bacteriostasis rate of ovotransferrin was 48.84%. [Conclusion] The procedure for separating lysozyme, ovotransferrin and ovalbumin from egg white was simple, fast, low-cost and suitable for industrilization.

β-谷甾醇对OVA诱导的过敏性哮喘大鼠模型中Th17/Treg免疫失衡的调节

目的:探讨β-谷甾醇对卵白蛋白(OVA)诱导的过敏性哮喘大鼠的干预作用及潜在作用机制。方法:SD雄性大鼠随机分为正常组(CON)、模型组(M)、阳性药地塞米松组(DEX,0.075 mg/kg)、β-谷甾醇组(Sit,50 mg/kg),采用腹腔注射OVA氢氧化铝溶液致敏,雾化吸入OVA激发建立过敏性哮喘大鼠模型。雾化激发前30 min灌胃给药,连续给药7 d后,检测大鼠咳喘指标、气管酚红排泌量;HE染色观察肺组织病理变化;流式细胞术检测大鼠原代肺细胞活性氧(ROS)、凋亡水平及外周血中Th17和Treg细胞比例;生化法检测大鼠肺组织中MDA含量及T-SOD、GSH-Px活力;ELISA检测Th17、Treg相关细胞因子(TNF-α、IL-4、IL-6、IL-17A、IL-35)水平。结果:与模型组相比,β-谷甾醇明显延长了过敏性哮喘大鼠的咳嗽及引喘潜伏期,减少了咳嗽及喘息次数,促进了气管酚红排泌;显著减轻了哮喘大鼠肺组织炎症浸润;显著降低了哮喘大鼠肺组织MDA含量、原代肺细胞ROS和凋亡水平,提高了T-SOD及GSH-Px活力;显著降低了Th17细胞比例及促炎细胞因子TNF-α、IL-4、IL-6、IL-17A水平,提高了Treg细胞比例及抑炎细胞因子IL-35水平。结论:β-谷甾醇能够改善OVA诱导的过敏性哮喘大鼠气道炎症和氧化损伤,其作用机制可能与β-谷甾醇对Th17/Treg免疫失衡及氧化应激反应的调节有关。

Protective effect of Bifidobacterium infantis CGMCC313-2 on ovalbumin-induced airway asthma and b-lactoglobulininduced intestinal food allergy mouse models

AIM To determine whether oral administration of Bifidobacterium infantis CGMCC313-2(B. infantis CGMCC313-2) inhibits allergen-induced airway inflammation and food allergies in a mouse model.METHODS Ovalbumin(OVA)-induced allergic asthma and b-lactoglobulin-induced food allergy mouse models were used in this study. Following oral administration of B. infantis CGMCC313-2 during or after allergen sensitization, histopathologic changes in the lung and intestine were evaluated by hematoxylin and eosin(HE) staining. In the allergic asthma mouse model, we evaluated the proportion of lung-infiltrating inflammatory cells. OVAspecific IgE and IgG1 levels in serum and cytokine levels in bronchoalveolar lavage fluid(BALF) were also assessed. In the food allergy mouse model, the levels of total Ig E and cytokines in serum were measured.RESULTS Oral administration of B. infantis CGMCC313-2 during or after allergen sensitization suppressed allergic inflammation in lung and intestinal tissues, while the proportion of infiltrating inflammatory cells was significantly decreased in the BALF of allergic asthma mice. Moreover, B. infantis CGMCC313-2 decreased the serum levels of total Ig E in food allergy mice, and reductions in IgE and IgG1 were also observed in OVA-induced allergic asthma mice. The expression of interleukin-4(IL-4) and IL-13 in both serum and BALF was suppressed following the administration of B. infantis CGMCC313-2, while an effect on serum IL-10 levels was not observed.CONCLUSION B. infantis CGMCC313-2 inhibits the secretion of allergen-induced IgE, IL-4 and IL-13, and attenuates allergic inflammation.

定喘颗粒对OVA诱导豚鼠急性哮喘的作用研究

目的探讨定喘颗粒对于卵清蛋白(Ovalbumin,OVA)诱导的豚鼠急性哮喘模型的影响。方法通过腹腔注射OVA溶液并进行行为学评分后,随机分为模型组、硫酸特布他林组、喘嗽宁片组、定喘颗粒低剂量组、定喘颗粒中剂量组、定喘颗粒高剂量组,并从未处理豚鼠中随机选取8只作为空白组。在给药2周后通过OVA喷雾进行刺激,检测模型组及各给药组中豚鼠的引喘潜伏期,利用酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)检测豚鼠血清和支气管肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)中白细胞介素-4(Interleukin-4,IL-4)和白细胞介素-10(Interleukin-10,IL-10)的水平,并借助嗜酸粒细胞直接计数液检测全血和BALF中嗜酸性粒细胞(Eosinophil,EOS)的数量,苏木素-伊红(Hematoxylin-eosin,HE)染色检测豚鼠肺部组织的病理变化情况并评估。结果与空白组相比,模型组炎症相关指标出现明显异常,指示造模成功。与模型组相比,阳性药组尤其是硫酸特布他林片组,以及定喘颗粒高剂量组给药均可延长OVA诱发急性支气管哮喘豚鼠的引喘潜伏期,降低血液及BALF中IL-4和IL-10的水平和EOS的数量,并缓解肺部组织的病理变化。结论定喘颗粒可以有效缓解急性哮喘时出现的炎症情况,并减少哮喘相关炎症导致的肺部病理损伤。

The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant-free condition

Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when QVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens.

Structural and functional properties of hydrolyzed/glycosylated ovalbumin under spray drying and microwave freeze drying

Ovalbumin(OVA),the main protein in egg white,affects most of the functional properties of egg white protein in food processing.The aim of this study was to investigate the effects of spray drying(SD)and microwave freeze drying(MFD)on the preparation of hydrolyzed/glycosylated ovalbumin(HGOVA)and provide useful information on the applications of egg protein powders in the food industry.Results demonstrated that the structure of HGOVA was considerably changed,and its functional properties were improved compared with those of native OVA.SD and MFD processing did not lead to dissociation of HGOVA subunits.SD-HGOVA exhibited higher protein solubility,emulsifying activity,foaming capacities,and gel hardness than MFD-HGOVA.However,MFD-HGOVA was better than the SD-HGOVA in terms of color,emulsion stability,foam stability,water/oil absorption capacity,and thermal stability.Selection of an appropriate drying method could enhance the potential applications of HGOVA in the food industry.

Electrochemical study of pyrite-ovalbumin interaction in relation to flotation

Ovalbumin （OVA）, chicken egg albumin, is an environmentally friendly, non-toxic, cheap surface active agent. It has electrochemically active sulfhydryl residues in molecular structure. OVA adsorption on pyrite as an alternative depressant was investigated by cyclic voltammetry, FTIR spectroscopy and flotation. Tests were conducted in a wide potential range in alkaline pH to clarify the role of electrochemical condition on the adsorption mechanisms and the rate of depression. In the absence of OVA, the floatability reached its maxima around slightly oxidizing condition. Beyond the range, it decreased presumably due to the formation of Fe-oxyhydroxides together with the hydrophilic S-containing species. OVA-pyrite interaction occurred in the whole examined potential range. Depressing effect of OVA increased gradually from reducing to slightly oxidizing potential, open circuit potential （OCP） of pyrite, mainly due to weak conformational changes in OVA molecules together with the hydrophobic interaction around OCP. The rate of depression reached its maxima at moderately oxidizing potentials, which was referred to electrostatic attraction. This level was almost kept at higher potentials owing to OVA adsorption as metal-chelates.

Preparation and Characterization of β-Cyclodextrin Derivatized Ovalbumin Used as Chiral Selector in Pressure Capillary Electrochromatography

Synthesis and properties of β-cyclodextrin derivatized ovalbumin used as chiral selector were investigated, β-cyclodextrin derivatized ovalbumin was synthesized using β-cyclodextrin and ovalbumin in the presence of ethylene glycol diglycidyl ether in boric acid buffer at pH value 8.7 at 37℃. Amino group was coated on the internal surface of the silica capillary by sol-gel technology with triethoxylmethylsiloxane and （3-aminopropyl）trimethoxysiloxane. Covalent binding of g-cyclodextrin derivatized ovalbumin was performed by glutaraldehyde. Enantiomers of chlorpheniramine, phenylalanine and atropine were separated by pressure capillary electrochromatography column coated with β-cyclodextrin derivatized ovalbumin.

Cyclodextrin/chitosan nanoparticles for oral ovalbumin delivery: Preparation, characterization and intestinal mucosal immunity in mice

A novel oral protein delivery system with enhanced intestinal penetration and improved antigen stability based on chitosan(CS) nanoparticles and antigen-cyclodextrin(CD) inclusion complex was prepared by a precipitation/coacervation method. Ovalbumin(OVA) as a model antigen was firstly encapsulated by cyclodextrin, either β-cyclodextrin( β-CD) or carboxymethyl-hydroxypropyl-β-cyclodextrin(CM-HP-β-CD) and formed OVA-CD inclusion complexes, which were then loaded to chitosan nanoparticles to form OVA loaded β-CD/CS or CM-HP-β-CD/CS nanoparticles with uniform particle size(836.3 and 779.2 nm, respectively) and improved OVA loading efficiency(27.6% and 20.4%, respectively). In vitro drug release studies mimicking oral delivery condition of OVA loaded CD/CS nanoparticles showed low initial releases at p H 1.2 for 2 h less than 3.0% and a delayed release which was below to 30% at p H 6.8 for further 72 h. More importantly, after oral administration of OVA loaded β-CD/CS nanoparticles to Balb/c mice, OVA-specific sIgA levels in jejunum of OVA loaded β-CD/CS nanoparticles were 3.6-fold and 1.9-fold higher than that of OVA solution and OVA loaded chitosan nanoparticles, respectively. In vivo evaluation results showed that OVA loaded CD/CS nanoparticles could enhance its efficacy for inducing intestinal mucosal immune response. In conclusion, our data suggested that CD/CS nanoparticles could serve as a promising antigen-delivery system for oral vaccination.

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

The LTB of enterotoxigenic Escherichia coli (ETEC) expressed in Bifidobacterium infantis (BI) has been testified as mucosal adjuvant with co-vaccination BI-CfaB (the major fimbrial subunit) together in vivo in our previous study. In order to investigate the mucosal adjuvant effect of BI-LTB to purified antigens, we oral vaccinated SD rats with recombinant BI-LTB plus OVA (rBI-LTB + OVA), and wild type BI plus OVA (wBI + OVA), OVA and PBS (Phosphate buffered saline) were vaccinated as controls, respectively. The OVA-specific sIgA in jejunal mucosa and specific IgG in serium were measured with ELISA (Enzyme-linked immunosorbent assay) and the sIgA producing cells were detected with immunohistochemistry technology (IHC) and Qwin image manipulation tools subsequently. The results shown rBI-LTB could stimulate SD rats produce high titer OVA-specific sIgA in rBI-LTB + OVA group and the OVA-specific sIgA titer in rBI-LTB + OVA group was found significant greater than that of the wBI + OVA group or OVA single group (p < 0.05). However no such significant difference was detected between the group wBI + OVA and OVA. IHC results suggested that intestinal mucosa and submucosa was the main field of sIgA secretion. These results suggested that recombinant LTB expression in BI could be used as a wide range mucosal adjuvant with different form antigens.