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.

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.

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 (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 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.

目的 构建多房棘球蚴抗原亮氨酸氨基肽酶(Leucine aminopeptidase,LAP)原核表达系统,并做纯化及最佳酶活性条件分析,为后续作用机制研究奠定基础.方法 利用OptimumTM Codon软件对LAP编码序列进行优化后合成目的基因,连接入表达载体pCznl后转化入大肠杆菌BL-21菌株,在发酵过程中加入IPTG诱导目的蛋白表达后,通过Ni柱亲和纯化获得高纯度重组目的蛋白LAP.以亮氨酸对硝基苯胺(Leu-pNA)为底物测定LAP酶活性,观察酶活性的最适pH和温度,以及不同金属离子对其酶活性的影响.结果 基因测序和酶切鉴定显示pCznl-LAP质粒构建成功,通过Ni柱亲和纯化得到一个大小约为57 KD的蛋白.当pH为9.0、温度为60℃时LAP酶活性最强;金属离子对LAP酶活性影响的小大顺序为Co2+>Zn2+>Ca2+>Mn2+>Mg2+>Ba2+>K+.结论 本研究成功构建LAP原核表达系统,表达和纯化了LAP蛋白,获得了酶活性最强时的基础条件,为研发抗寄生虫药物及探究LAP在多房棘球蚴感染过程的作用机制奠定了基础.

目的 评价重组抗原蛋白14-3-3的免疫原性和其在多房棘球绦虫原头节继发性感染BALB/c小鼠模型中的预防及治疗效果.方法 构建多房棘球绦虫重组抗原蛋白14-3-3的原核表达系统,经IPTG诱导和Ni2+亲和层析法得到高纯度的14-3-3重组蛋白免疫BALB/c小鼠.用ELISA法检测免疫后小鼠血清中的抗体和细胞因子水平变化;在泡球蚴小鼠继发性感染模型中观察囊泡数量和重量.结果 成功表达并获取高纯度的多房棘球绦虫重组14-3-3抗原蛋白,免疫原性研究发现重组抗原蛋白14-3 3具有较好的免疫原性并激活小鼠产生Thl/Th2混合型免疫应答;在预防效果评价模型中,经14-3-3抗原免疫后,小鼠囊泡数量减少(59.84±15.09％),P=0.049,囊泡重量减轻(83.28±18.01％),P=0.000;在治疗效果评价模型中,经14-3-3抗原免疫后小鼠囊泡重量减轻(72.28±11.47％),P=0.006.结论 多房棘球绦虫重组抗原蛋白14-3-3对继发性小鼠泡球蚴病具有预防和治疗作用.