BACKGROUND:Conventional monoclonal antibodies generated by hybridoma technology have long been applied in disease diagnosis and treatment, yet they are constrained by inherent limitations, including epitope loss caused by chemical antigen processing and insufficient tissue penetration. Nanobodies, as single-domain antibody fragments derived from camelids and chondrichthyans, possess unique superior physicochemical characteristics such as low molecular weight, low immunogenicity, easy modification, strong tissue permeability, and precise epitope targeting. Notably, camelid-derived nanobodies exhibit high homology with human antibody subfamilies and favorable biosafety, showing promising potential for biomedical applications. METHODS:This review systematically summarizes the unique structural properties and technical advantages of nanobodies. It comprehensively sorts out the latest research progress of nanobodies in common respiratory diseases, including asthma, tuberculosis, respiratory infections, and lung cancer, and further analyzes their unique superiority in pulmonary drug delivery and clinical transformation. RESULTS:Nanobodies retain the specific antigen-binding capacity equivalent to traditional monoclonal antibodies while overcoming the defects of conventional antibody preparations. Their excellent modifiability, tissue penetration and low immunogenicity enable them to achieve accurate targeted binding in respiratory tissues. Accumulating evidence verifies the outstanding diagnostic performance and therapeutic efficacy of nanobodies in multiple respiratory disorder models, providing new technical support for pulmonary drug delivery and precise intervention of respiratory diseases. CONCLUSION:Nanobodies possess irreplaceable advantages in the diagnosis and treatment of respiratory diseases and have broad clinical translation prospects. This review clarifies the application value of nanobodies in mainstream respiratory diseases and proposes innovative strategies for the precise diagnosis and targeted treatment of respiratory disorders, laying a theoretical foundation for further clinical transformation and in-depth application of nanobody-based respiratory therapeutics.

Mosquito-borne diseases (MBDs) are a major global public health concern, accounting for 17% of infectious diseases and causing ~700,000 annual deaths. Transmitted by Aedes, Anopheles, and Culex mosquitoes, they include viral (dengue, Zika, chikungunya), parasitic (malaria, lymphatic filariasis), and zoonotic (Japanese encephalitis) pathogens. This review outlines key MBDs’ clinical features, global distribution—concentrated in tropics but expanding due to climate change and urbanization—and transmission dynamics shaped by environmental (temperature, humidity) and ecological (urban breeding sites) factors. It also summarizes control strategies: vector management, vaccines (e.g., R21 for malaria, IXCHIQ for chikungunya), chemoprophylaxis, and novel technologies. Moreover, persistent challenges are covered, which include insecticide resistance and socioeconomic costs (e.g., $318 billion for Aedes-borne diseases since 1975), emphasizing the need for integrated interventions.

[This corrects the article DOI: 10.3389/fimmu.2025.1683003.].

BackgroundAlveolar Echinococcosis (AE) is a serious infectious disease caused by Echinococcus multilocularis (E.multilocularis,Em) in the highlands of northwestern China and vaccination is currently the most effective means of preventing E. multilocularis infection. However, current vaccines are not sufficiently effective in preventing and controlling Alveolar Echinococcosis.MethodsIn this study, an oral M-cell targeted Lactococcus lactis (L. lactis) vaccine (LL-plSAM-GILE) was constructed by adding SAM gene sequence to the epitope vaccine GILE for E. multilocularis constructed in our previous study. Mice were orally immunized with LL-plSAM-GILE and their serum antibody levels (ELISA), lymphocyte proliferation (MTS), IFN-γ levels (ELISpot), IL-4 levels (flow cytometry, FCM), T cells (FCM), growth of hepatic cysts (Ultrasound), and weights were measured to evaluate the protective effect of LL-plSAM-GILE.ResultsThe L.lactis expression plasmid pNZ8148-SAM-GILE was successfully constructed and electroporated into L.lactis NZ9000, and the recombinant protein was approximately 45 KD. SAM-GILE was expressed on the surface of recombinant L.lactis. LL-plSAM-GILE is effective in targeting Microfold cells. Mice immunized with LL-plSAM-GILE exhibited significantly elevated levels of specific IgG antibodies. Lymphocyte proliferation was enhanced compared to the control group and the NZ9000 group. LL-plSAM-GILE stimulated the production of CD4+ and CD8+ T cells. Mice immunized with LL-plSAM-GILE secreted more IFN-γ and IL-4. For both primary and secondary infections, oral immunization with LL-plSAM-GILE led to a significant decrease in the diameter and weight of hepatic cysts.ConclusionsAn oral M-cell targeted L.lactis vaccine LL-plSAM-GILE with excellent immunogenic and immunoprotective properties has been successfully constructed. This study may provide important theoretical and experimental bases for the prevention and treatment of E. multilocularis infection.

BackgroundCystic echinococcosis (CE), caused by Echinococcus granulosus, is a zoonotic disease with major global social and economic impacts. Research on its burden in children and adolescents remains limited. This study evaluates the global CE burden from 1990 to 2021 and projects future trends, supporting WHO NTD Roadmap goals aimed at enhancing control in 17 high-endemic countries by 2030.MethodsUsing the Global Burden of Disease (GBD) database, we assessed prevalence, incidence, deaths, DALYs, YLDs, and YLLs due to CE in individuals aged 0-19 at global, regional, and national levels. We computed age-standardized rates (ASRs) and estimated annual percentage changes (EAPCs). Additional analyses included joinpoint regression, inequality measures, frontier and decomposition analysis, age-period-cohort (APC) modeling, Socio-demographic Index (SDI) correlations, and future trend prediction.ResultsOver 32 years, the global CE burden declined overall, though disparities persisted. Low SDI regions had high ASPR, ASIR, and ASMR. In 2021, global ASIR was 1.12 per 100,000, ASPR was 3.71, and ASMR was 0.01. Moldova had the highest ASPR; Iceland the lowest. East Asia saw growth in ASPR and ASIR. South Sudan had the highest ASMR; Ethiopia had the highest ASDR. Females showed higher ASPR and ASIR; males had higher ASMR. A strong negative correlation was observed between SDI and health indicators. Population changes primarily influenced ASPR. Frontier analysis indicated elevated ASMR/ASDR in some low-SDI nations and rising trends in certain high SDI countries. Age-specific prevalence increased with age. Projections suggest a slow decline in CE burden over the next 25 years, though some countries will remain severely affected.ConclusionsThe global CE burden in children and adolescents decreased from 1990 to 2021, yet challenges remain, especially in low-SDI regions such as Sub-Saharan Africa and Central Asia. The Slope Index of Inequality (SII) for ASPR narrowed from -2.597 to -1.087, reflecting reduced but persistent disparity. Rising ASMR and ASDR in high SDI countries like Germany and Norway underscore the need for targeted interventions. The negative SDI health correlation highlights socioeconomic influences. Prevention should focus on females in low-SDI areas, while improved medical care is needed for males facing higher mortality. Although a continued decline is projected, sustained efforts are essential in high burden countries. These findings, supported by a improving concentration index (CI) for ASPR (-0.358 to -0.218), reveal critical health inequalities and inform public health strategies.

目的 构建幽门螺旋杆菌尿素酶纳米抗体UreNb重组质粒,在大肠杆菌系统中通过诱导表达目的蛋白并予以纯化,再对表达产物及纯化产物进行鉴定.方法 利用DNA重组技术,先后使用两种原核表达载体pET22b和pSumo-mut表达目的蛋白UreNb,构建幽门螺旋杆菌尿素酶纳米抗体UreNb,重组质粒.将构建成功的纳米抗体UreNb重组质粒分别转化至大肠杆菌BL-21、Arctic Express、Rosetta菌株中,采用不同温度经IPTG诱导表达目的蛋白.将成功表达的重组蛋白扩大培养,通过SDS-PAGE法分析其表达蛋白,表达蛋白在沉淀物中即确定为包涵体蛋白.将包涵体蛋白变、复性后,通过镍柱亲和层析法纯化蛋白并用 Western Blot法鉴定.结果 纳米抗体UreNb目的序列成功插入质粒中,在大肠杆菌表达系统BL-21 中以包涵体形式表达(经SDS-PAGE法验证).经过包涵体复性后纯化,获得大量高纯度目的蛋白pSumo-mut-UreNb,纯化蛋白的分子量约为 27 kDa(经Western Blot鉴定).结论 使用pSumo-mut质粒构建的幽门螺旋杆菌尿素酶纳米抗体UreNb能够在原核系统中得到高效表达,通过纯化获得具有活性的纯化蛋白.经鉴定,其大小与理论大小基本相符,可用于后续研究.

目的 探究放血疗法是否参与了对慢性低氧环境下雄性大鼠红细胞衰亡的调控.方法 低氧组(Hypoxia)和放血组(Phlebotomy)雄性大鼠置模拟海拔 5 000 m的低压氧舱 28 d,Phle-botomy分别于第7、14、21 d经尾静脉放血500 μL进行动态血常规分析.4 w后取血进行血细胞分析、渗透脆性检验、红细胞凋亡检测、网织红细胞检测等.结果 放血后雄性大鼠红细胞数量和血红蛋白含量结果:Hypoxia(10.58±0.63)与Phlebotomy(9.58±0.31),Hypoxia(174.50±7.87)与Phlebotomy(161.17±4.40),组间差异有统计学意义(P=0.004,P=0.037).放血后红细胞比容结果:Hypoxia(61.36±2.21)与Phlebotomy(56.73±1.70),组间差异有统计学意义(P=0.001).流式细胞术分析红细胞衰亡:Hypoxia(0.07±0.01)与Phlebotomy(0.10±0.02),组间差异有统计学意义(P=0.001).红细胞衰亡动态结果:2 w(0.20±0.01)与3 w(0.15±0.02),3 w(0.15±0.02)与 4 w(0.09±0.02),组间差异有统计学意义(P=0.000,P=0.000).网织红细胞动态结果:1 w(0.04±0.03)、2 w(0.05±0.04)、3 w(0.04±0.02)、4 w(0.06±0.03)组间差异无统计学意义.结论 放血疗法可促进高原慢性低氧下雄性大鼠红细胞的衰亡,降低慢性低氧对雄性大鼠红细胞衰亡的抑制作用.同时放血不会持续增加雄性大鼠红细胞衰亡,也不会持续刺激雄性大鼠网织红细胞增加.

Introduction The objective of this study is to construct a multi-epitope vaccine GILE containing B-cell and T-cell epitopes against Echinococcus Multilocularis (E. multilocularis) infection based on the dominant epitopes of E. multilocularis EMY162, LAP, and GLUT1. Methods The structure and hydrophobicity of GILE were predicted by SWISSMODEL, pyMOL, SOPMA and VMD, and its sequence was optimized by Optimum™ Codon. The GILE gene was inserted into pCzn1 and transformed into Escherichia coli Arctic express competent cells. IPTG was added to induce the expression of recombinant proteins. High-purity GILE recombinant protein was obtained by Ni-NTA Resin. BALB/c mice were immunized with GILE mixed with Freund’s adjuvant, and the antibody levels and dynamic changes in the serum were detected by ELISA. Lymphocyte proliferation was detected by MTS. The levels of IFN-g and IL-4 were detected by ELISpot and flow cytometry (FCM). T cells were detected by FCM. The growth of hepatic cysts was evaluated by Ultrasound and their weights were measured to evaluate the immune protective effect of GILE. Results The SWISS-MODEL analysis showed that the optimal model was EMY162 95-104―LAP464-479―LAP495-510―LAP396-410―LAP504-518―EMY162112-126. The SOPMA results showed that there were Alpha helix (14.88%), Extended strand (26.25%), Beta turn (3.73%) and Random coil (45.82%) in the secondary structure of GILE. The restriction enzyme digestion and sequencing results suggested that the plasmid pCzn1-GILE was successfully constructed. The SDSPAGE results indicated that the recombinant protein was 44.68 KD. The ELISA results indicated that mice immunized with GILE showed higher levels of serum antibodies compared to the PBS group. The FCM and ELISpot results indicated that mice immunized with GILE secreted more IFN-g and IL-4. Immunization with GILE also led to a significant decrease in the maximum diameter and weight of cysts and stimulated the production of CD4+ and CD8+ T Cell. Discussion A multi-epitope vaccine GILE with good immunogenicity and antigenicity has been successfully constructed in this study, which may provide important theoretical and experimental bases for the prevention and treatment of E. multilocularis infection.

BACKGROUND: Alveolar echinococcosis is an epidemic disease caused by the parasitism of Echinococcus multilocularis (Em) larvae in the intermediate or final host. OBJECTIVE: To identify and analyze B-cell and T-cell (Th1, Th2, and Th17) epitopes of the Em antigen protein thrombospondin 3 (TSP3). METHODS: The amino acid sequence of TSP3 was obtained, and the secondary structural characteristics of TSP3 were predicted using bioinformatics software to further predict its potential T-cell and B-cell epitopes. The spleen lymphocytes of BALB/c mice, which were immunized with the TSP3 protein, were collected for co-culture with B-cell and T-cell antigen small peptides. The B-cell epitopes and T-cell epitope subtypes Th1, Th2, and Th17 were identified as having good immunogenicity. RESULTS: After identification, it was found that the predominant epitopes of B cells existing in TSP3 were T18-33, T45-55, and T110-122. Furthermore, the predominant epitopes of T cells existing in TSP3 were T33-42, T45-55, T80-90, and T110-122 in the T1 subtype, T45-55, T68-77, and T92-104 in the Th2 subtype, and T53-63 and T80-90 in the Th17 subtype. CONCLUSIONS: Six T-cell and eight B-cell dominant epitopes of the TSP3 antigen were revealed; these results may be applied in the development of a dominant epitope vaccine. Keywords Echinococcus multilocularis , bioinformatics , epitopes

BACKGROUND: Alveolar echinococcosis is an epidemic disease caused by the parasitism of Echinococcus multilocularis (Em) larvae in the intermediate or final host. OBJECTIVE: To identify and analyze B-cell and T-cell (Th1, Th2, and Th17) epitopes of the Em antigen protein thrombospondin 3 (TSP3). METHODS: The amino acid sequence of TSP3 was obtained, and the secondary structural characteristics of TSP3 were predicted using bioinformatics software to further predict its potential T-cell and B-cell epitopes. The spleen lymphocytes of BALB/c mice, which were immunized with the TSP3 protein, were collected for co-culture with B-cell and T-cell antigen small peptides. The B-cell epitopes and T-cell epitope subtypes Th1, Th2, and Th17 were identified as having good immunogenicity. RESULTS: After identification, it was found that the predominant epitopes of B cells existing in TSP3 were T18-33, T45-55, and T110-122. Furthermore, the predominant epitopes of T cells existing in TSP3 were T33-42, T45-55, T80-90, and T110-122 in the T1 subtype, T45-55, T68-77, and T92-104 in the Th2 subtype, and T53-63 and T80-90 in the Th17 subtype. CONCLUSIONS: Six T-cell and eight B-cell dominant epitopes of the TSP3 antigen were revealed; these results may be applied in the development of a dominant epitope vaccine.

Introduction: The main objective of this study was to develop a One-tube nested MGB probe real-time PCR Assay for detecting Echinococcus multilocularis infection in human plasma cell free DNA (cfDNA). Methods: cfDNA was extracted from 10 E.m.-infected patients using a NucleoSnap DNA Plasma Kit and characterized by genomic sequencing. We designed nested PCR primers and MGB probe for Echinococcus multilocularis detection. The specificity, sensitivity and reproducibility of this assay were analyzed, and its validity was confirmed in 13 early stage clinical samples. Results: Several Echinococcus multilocularis-specific sequences were detected in the cfDNA of E.m.-infected patients, and CBLO020001206.1 was selected as the candidate sequence. We designed the primers and probe for the one tube nested real-time PCR. No cross-reactions with E.g. were observed. The detection limit was as low as 1 copy for Echinococcus multilocularis. The coefficients of variation were lower than 5% in intra- and inter-assays. 11 out of 13 patients were positive with nested MGB Probe PCR Assay and 3 patients were positive without outer primer in early stage Alveolar Echinococcosis pateints. Conclusion: The one-tube nested MGB probe real-time PCR assay is a simple, rapid, and cost-effective method for detection of Echinococcus multilocularis infection in patients' Plasma DNA.

目的 通过对多房棘球蚴葡 萄糖转 运蛋白(Echinococcus multilocularis glucose transporter,EmGLUT)二级结构的分析,并结合在线工具来预测优势T细胞和B细胞抗原表位.方法 从NCBI-Gen Bank库中获取EmGLUT基因的mRNA和氨基酸序列.二级结构特征预测使用SOPMA软件.使用IEDB、Syfpeithi软件分析T细胞优势抗原表位,使用Bcepred、AB-Cpred软件分析B细胞优势抗原表位.结果 分析结果显示,在EmGLUT二级结构中α螺旋比例占51.08％,β折叠比例占17.88％,β转角比例占3.73％,无规则卷曲比例占27.31％.综合5个在线软件分析结果得出EmGLUT有10个T细胞优势抗原表位和18个B细胞优势抗原表位,其中5个同时具有T-B细胞双抗原表位.结论 EmGLUT的优势T细胞和B细胞抗原表位被成功预测.

There are many virulence factors of H. pylori that contribute in diverse ways to gastric disease. Therefore, designing multivalent epitope vaccines against many key virulence factors virulence factors of H. pylori is a promising strategy to control H. pylori infection. In previous studies, we constructed a multivalent epitope vaccine FVpE against four key virulence factors of H. pylori (Urease, CagA, VacA, and NAP), and oral immunization with the FVpE vaccine plus a polysaccharide adjuvant (PA) containing lycium barbarum polysaccharide and chitosan could provide protection against H. pylori infection in the Mongolian gerbil model. Oral vaccines have many advantages over injected vaccines, such as improved safety and compliance, and easier manufacturing and administration. However, the harsh gastrointestinal (GI) environment, such as gastric acid and proteolytic enzymes, limits the development of oral vaccines to some extent. Oral vaccines need a gastrointestinal delivery system with high safety, low price and promoting vaccine antigen to stimulate immune response in the gastrointestinal mucosa. Lactic acid bacteria are gastrointestinal probiotics that have unique advantages as a delivery system for oral vaccines. In this study, a M cell-targeting surface display system for L. lactis named plSAM was designed to help vaccine antigens to stimulate effective immune responses in the gastrointestinal tract, and a M cell-targeting recombinant L. lactis vaccine LL-plSAM-FVpE was constructed by using the surface display system plSAM. recombinant L. lactis vaccine LL-plSAM-FVpE could secretively express the SAM-FVpE protein and display it on the bacterial surface. Moreover, experimental results confirmed that LL-plSAM-FVpE had an enhanced M cell-targeting property. In addition, LL-plSAM-FVpE had excellent M cell-targeting property to promote the phagocytosis and transport of the antigen SAM-FVpE by gastrointestinal M cells. More importantly, oral immunization of LL-plSAM-FVpE or SAM-FVpE plus PA can stimulate IgG and sIgA antibodies and CD4+ T cell immune responses against four virulence factors of H. pylori (Urease, CagA, VacA, and NAP), thus providing protective immunity against H. pylori infection in mice. The M cell-targeting recombinant L. lactis vaccine against various key H. pylori virulence factors could be a promising vaccine candidate for controlling H. pylori infection.

目的 设计、构建多房棘球绦虫多表位疫苗GILE,并表达、纯化该重组蛋白.方法 基于课题组前期对多房棘球绦虫EmEMY162、EmLAP、EmGLUT1优势抗原表位鉴定的结果,通过SWISS-MODEL、pyMOL、SOPMA、VMD生物信息学软件预测GILE的结构及疏水性,设计多表位疫苗GILE.合成GILE序列,插入质粒pCzn1中,构建重组质粒pCzn1-GILE.将重组质粒转化入大肠杆菌Artic express中,添加IPTG诱导重组蛋白表达.通过SDS-PAGE和免疫印迹法确定重组蛋白是否表达.结果 通过SWISS-MODEL软件开展同源建模,结果表明抗原表位最优串联方式为EMY16295-104—LAP464-479—LAP495-510—LAP396-410—EMY162106-121—LAP504-518—EMY162112-126.通过SOPMA软件分析GILE的二级结构,结果表明α螺旋占13.84％,β折叠占26.25％,β转角占14.88％,无规卷曲占45.82％.通过酶切和测序验证表明,质粒pCzn1-GILE构建成功.通过IPTG诱导,得到45KD的重组蛋白.通过Ni-NTA柱的亲和纯化,得到纯化重组蛋白GILE.通过West-ern blotting法验证,纯化后的GILE蛋白能与His抗体特异结合,得到纯化重组蛋白GILE.结论 本研究通过生物信息学软件成功设计、构建了多表位疫苗GILE,并通过GILE原核表达系统表达、纯化了重组蛋白GILE.

Alveolar Echinococcosis is a globally widespread zoonotic disease caused by the larval stage of Echinococcus multilocularis (Em) and is seriously harmful to human health. In our previous studies, we found that Em-EMY162 had good prophylactic and therapeutic effects against Em infection and the dominant epitopes of Em-EMY162 and Em-TSP3 were also identified. In this study, a multi-epitope vaccine LTB-ETBM targeting both Em-EMY162 and Em-TSP3 was designed and constructed, and then the immunogenicity and protective efficacy were evaluated in Em infected mice. The results showed that LTB-ETBM could induce high levels of specific IgG against Em-EMY162 and Em-TSP3 and a mixed Th1/Th2 lymphocyte response to LTB-ETBM. Moreover, the LTB-ETBM significantly inhibited the formation of cysts in mice challenged with 1000 Em protoscoleces. In a therapeutic mouse model injected intraperitoneally with 1000 protoscoleces, vaccination with LTB-ETBM using either Freund's or CpG as an adjuvant significantly decreased the growth of protoscoleces and the formation of cysts. LTB-ETBM may be efficacious for use as a prophylactic or therapeutic agent against Em infection.

Alveolar echinococcosis (AE) is a parasitic disease caused by E. multilocularis metacestodes and it is highly prevalent in the northern hemisphere. We have previously found that vaccination with E. multilocularis Leucine aminopeptidase (EM-LAP) induced specific immune response and had an inhibiting effect on the parasites. In this study, the therapeutic effect of recombinant EM-LAP (rEM-LAP) on AE was evaluated and verified using Ubenimex, a broad-spectrum inhibitor of LAP. The results reveal that rEM-LAP could inhibit cyst growth and invasion and induce specific immunity response in BALB/c mice infected with E. multilocularis protoscoleces. The ultrasonic, MRI, and morphological results show that treatment with rEM-LAP inhibits E. multilocularis infection and reduces cyst weight, number, fibrosis and invasion. The same effect is observed for the treatment with Ubenimex by inhibiting LAP activity. The indirect ELISA shows that rEM-LAP could induce specific immunity response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA, and the serum levels of IFN-γ and IL-4 are significantly increased compared to the control groups, indicating that treatment with rEM-LAP leads to a Th1 and Th2 mixed-type immune response. This study suggests that EM-LAP could be a potential therapeutic target of E. multilocularis infection.

Alveolar echinococcosis, a parasitic zoonotic disease caused by the larval stage of Echinococcus multilocularis infection, is a global epidemic in Eurasia and North America. Leucine aminopeptidase (LAP) of the M17 peptidase family could act on an ideal target antigen in diagnosis and prevention of parasitic diseases (schistosomiasis, malaria, fascioliasis) because of its good immunogenicity. In this study, the bioinformatic and enzymatic characterizations of recombinant Echinococcus multilocularis LAP (rEm-LAP) were evaluated. A prokaryotic expression system for rEm-LAP protein was established and its immunogenicity and preventive efficacy were demonstrated in a BALB/c mice model. This is the first report about the LAP of Echinococcus multilocularis and with a 57.4 KD purified rEm-LAP protein successfully expressed by pCzn1-LAP in Escherichia coli BL-21 cells. Enzymatic analysis results showed optimal rEm-LAP activity at pH 9. Serum indirect ELISA demonstrated that rEm-LAP could induce a Th1 and Th2 mixed-type immunological response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA. Furthermore, serum IFN-γ and IL-4 secretion were increased compared with the control groups. Finally, vaccination with rEm-LAP significantly decreased both the number and size of the cysts in Echinococcus multilocularis metacestode infected mice model. The current study provides evidence that rEm-LAP could be a potential vaccine antigen of Echinococcus multilocularis.

INTRODUCTION:Alveolar echinococcosis is a high-risk parasitic disease caused by the larval stage of Echinococcus multilocularis. The study aimed to predict and identify the dominant Th1/Th2 and B cell epitopes of the antigen protein 14-3-3 beta:alpha from Echinococcus multilocularis.METHODS:A comparison of the four amino acid sequences of 14-3-3 beta:alpha was respectively derived from Echinococcus multilocularis, Rattus norvegicus, Canis lupus familiaris, and Homo sapiens was carried out by CLUSTALW to provide a basis for excluding similar epitopes. The amino acid sequence information was analyzed by SOPMA and the homology model was established by Swiss-Model. IEDB and SYFPEITHI were used to predict T cell epitopes. According to the Bcepred and ABCpred, the B cell epitopes were comprehensively predicted and analyzed. The dominant epitopes were validated by Lymphocyte Proliferation, ELISA, ELISpot, and Flow cytometry.RESULTS:Eight potential epitopes of 14-3-3 from Echinococcus multilocularis were screened according to the results of prediction and analysis: 14-3-31-15, 14-3-36-21, 14-3-371-86, 14-3-3144-157, 14-3-3145-166, 14-3-3146-160, 14-3-3153-161, and 14-3-3164-177. The 3D structure model of the protein was constructed and the location distribution of potential epitope was ascertained. Respectively, the epitopes of the dominant antigen of B cells were validated as 14-3-3145-166 and 14-3-3164-177; the Th1 dominant antigen epitopes were 14-3-36-21, 14-3-3145-166; and the Th2 dominant epitopes was 14-3-3145-166.CONCLUSION:In this study, two dominant antigen epitopes of B cells, two Th1 dominant antigen epitopes, and one Th2 dominant antigen epitope were validated. Our work provides a basis for the subsequent development of efficient and safe vaccines targeting epitopes of Echinococcus multilocularis.

目的 设计构建及表达纯化多房棘球绦虫葡萄糖转运蛋白1重组抗原肽(GLEP),为后续疫苗研究寻找有效工具.方法 用生物信息学软件预测EmGLUT1跨膜结构,将设计构建的表达载体pC zn1-GLEP转化入大肠杆菌真核表达系统,经IPTG诱导、Ni-NTA纯化获得目的 蛋白.结果 生物信息学软件分析结果表明EmGLUT1为12次跨膜蛋白,用柔性序列GS和KK将筛选后的肽段进行串联,构建重组表达载体pCzn1-GLEP,测序及酶切验证成功后,转化入Artic ex-press大肠杆菌表达系统(表达部位鉴定属于可溶性表达),经Ni-NTA亲和柱层析纯化后获得电泳纯级的GLEP蛋白.结论 成功构建GLEP的原核表达系统,并得到纯化重组蛋白,为后续疫苗研究提供有效工具.

目的 根据预测多房棘球蚴亮氨酸氨基肽酶(Leucine aminopeptidase,LAP)抗原表位的结果,进一步鉴定其优势的T细胞和B细胞抗原表位.方法 构建重组质粒pCzn1-LAP后进行蛋白原核表达与纯化,将纯化的LAP蛋白进行动物免疫后,利用脾脏淋巴细胞增殖、流式细胞术、ELISA pot、ELISA等方法鉴定优势抗原表位.最后,利用SWISS-MODEL软件对LAP蛋白3D结构建模,PyMOL软件对鉴定的LAP优势抗原表位分布进行准确定位.结果 pCzn1-LAP菌株诱导表达的蛋白约为57 kDa,蛋白经过复性后成功纯化.流式细胞术鉴定的Th1型优势抗原表位为LAP79-63、LAP396-410和LAP106-120;Th2型优势抗原表位为LAP13-28、LAP495-510和LAP396-410.ELISA pot鉴定的Th1型优势抗原表位为LAP396-410、LAP504-518和LAP106-120;Th2型优势抗原表位为LAP504-518、LAP313-328和LAP396-410.BALB/c小鼠血清的抗原表位特异性ELISA筛选出的优势B表位结果为LAP464-479、LAP495-510和LAP313-328.LAP蛋白3D结构建模成功,LAP优势抗原表位分布于表面居多.结论 多房棘球蚴LAP蛋白优势T细胞和B细胞抗原表位鉴定成功,使得研制抗多房棘球蚴感染的多价表位疫苗成为可能.