目的研究豆状囊尾蚴丝氨酸蛋白酶抑制剂（Cpserpin）的组织分布及其对丝氨酸蛋白酶的抑制效果。方法利用原核表达的重组Cpserpin （rCpserpin）免疫家兔,采用间接ELISA检测免疫后血清的效价,取免疫兔血清,制备抗体并纯化后,采用Western blotting对多克隆抗体进行特异性分析,通过免疫组化试验分别观察rCpserpin在豆状带绦虫成虫和幼虫中的组织分布。构建重组质粒pPIC9K-Cpserpin,线性化后转化至毕赤酵母KM71感受态细胞,用G418筛选的高拷贝菌株进行诱导表达并纯化rCpserpin,采用发色底物法检测不同浓度（6、9、 18、 21μg/ml） rCpserpin对0.5μg/mlα-糜蛋白酶、胰蛋白酶和弹性蛋白酶等3种丝氨酸蛋白酶活性的抑制效果。结果间接ELISA检测结果显示,免疫血清多抗IgG抗体的效价达1∶51 200。Western blotting分析结果显示,纯化后的多克隆抗体可与rCpserpin和兔豆状囊尾蚴虫体可溶性抗原产生特异性反应。免疫组化试验结果显示,Cpserpin在囊尾蚴阶段表达量很低,但在成节和孕节中高度表达,广泛分布于虫体体壁和虫卵中。酵母表达的rCpserpin随浓度增加对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶的抑制效果分别增强,在21μg/ml浓度时,对3种丝氨酸蛋白酶的抑制率最高,分别为（50.5±2.5）%、（71.5±1.5）%和（77.4±1.5）%（均P ＜0.05）。结论 rCpserpin多克隆抗体具有特异性,在豆状囊尾蚴成节和孕节中高表达,且其对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶有较强的抑制作用。

Insulin signaling pathway is an ancient and highly conserved pathway known to play critical roles in cell growth, control and metabolic regulation. In this study, we identified and characterized two insulin receptor genes (TsIR-1316 and TsIR-4810) from Taenia solium. TsIR-1316 was grouped with E. multilocularis insulin receptor (EmIR-1) and TsIR-4810 was closer to Taenia pisiformis insulin-like growth factor receptor (TpIR) on the same branch with a very high bootstrap value. TsIR-1316 was located on the integument of larvae and adult worms, as well as the ovary of adults and eggs. Alternatively, TsIR-4810 was located in the parenchyma and reproductive organs of the adult worms. By using in vitro cultivation systems with Cysticercus pisiformis as a model, we demonstrated that anti-TsIRs-LBD antibodies could effectively block the insulin signaling pathway, resulting in reduced phosphorylation of the insulin receptor as well as lower levels of glucose uptake and glycogen synthesis. The rabbits immunized with TsIR-1316-LBD, TsIR-4810-LBD and TsIR-1316-LBD + TsIR-4810-LBD produced protection against infection of T. pisiformis as demonstrated by a 94.6%, 96% and 80% reduction of establishment of larvae, respectively. These data suggested that TsIR-1316-LBD and TsIR-4810-LBD are promising vaccine candidates or novel drug targets against swine cysticercosis.

目的 筛选并制备亚洲带绦虫45 kDa雌激素调节蛋白(TaEP45)的杂交瘤细胞株. 方法 设计带有酶切位点的特异性引物,以亚洲带绦虫成虫总RNA为模板,RT-PCR扩增TaEP45完整的开放阅读框(ORF),克隆至原核表达载体pET-30a(+),转化至大肠埃希菌BL21 (DE3),用1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达带组氨酸(His)标签的TaEP4蛋白,用亚洲带绦虫囊尾蚴阳性猪血清蛋白质印迹(Westernblotting)分析TaEP45的反应原性.以纯化的TaEP45免疫BALB/c小鼠(100 μg/鼠,首次免疫用等量弗氏完全佐剂,第2、第3次免疫用等量弗氏不完全佐剂),选择血清抗体效价高的小鼠,取脾细胞和SP2/0骨髓瘤细胞融合,用纯化的TaEP45和His标签蛋白ELISA筛选阳性杂交瘤细胞株,用亚洲带绦虫全虫抗原Western blotting鉴定杂交瘤细胞株分泌的单克隆抗体. 结果 RT-PCR扩增获得的TaEP45基因ORF长1 362 bp,编码453个氨基酸.重组融合蛋白的相对分子质量(Mr)约为60 000,可被亚洲带绦虫囊尾蚴阳性猪血清识别.筛选获得5株稳定分泌TaEP45抗体的杂交瘤细胞株,其抗体效价均≥1∶2 430,亚型分别为IgG2b或IgG1.Western blotting分析结果显示,5株杂交瘤细胞株分泌的单克隆抗体均能与亚洲带绦虫全虫抗原发生反应. 结论 表达纯化了TaEP45,筛选出5株能稳定分泌高滴度TaEP45单克隆抗体的杂交瘤细胞株,其分泌的单克隆抗体能与亚洲带绦虫全虫抗原发生反应.

Taenia asiatica is an important food-borne parasite that poses a threat to food-safety and animal husbandry hygine, yet little is known about its specific infection and immune escape mechanisms. Exosome-like vesicles have recently emerged as a regulator in the interactions between parasites and hosts, providing a new direction for research on infection of T. asiatica. In this experiment, exosome-like vesicles were collected from the excretory/secretory products of cultured T. asiatica and isolated by differential centrifugation. The purified vesicles, ranging from 30 to 150 nm in size, were identified as exosome-like vesicles by transmission electron microscope and Nanoparticle tracking analysis. Proteomics analysis identified 455 proteins in the exosome-like vesicles. Of these proteins, enzymes involved in metabolic processes were identified, including glyceraldehyde 3 phosphate dehydrogenase, fructose-1, 6-bisphosphate aldolase, cytosolic malate dehydrogenase, and enolase. The two most abundant proteins from proteomic analysis, 14-3-3 and enolase, were shown to be present in the exosome-like vesicles by immunogold labeling. High-throughput RNA sequencing yielded twenty known miRNAs present in exosome-like vesicle sRNA libraries. Nine of the miRNAs, including six known miRNAs (tas-miR-71, tas-miR-1, tas-miR-7, tas-miR-9, tas-miR-10, and tas-let-7) and three newly discovered miRNAs (tas-m0022-3p, tas-m0816-3p, tas-m0082-5p), were confirmed by RT-qPCR as present in T. asiatica adult worm extracts and secreted exosome-like vesicles in T. asiatica. Additionally, we demonstrated that exosome-like vesicles experimentally labeled with PKH67 were internalized by LoVo cells in vitro. These findings provide new insights into the interaction between tapeworms and hosts mediated by exosome-like vesicles.

为检测兔豆状囊尾蚴(Cysticercus pisiformis感染,利用cDNA末端快速扩增技术(RACE)扩增豆状囊尾蚴serpin基因,并对其开放阅读框(ORF)及编码的氨基酸序列进行生物信息学分析.构建pET-30a(+)-Cpserpin重组表达载体,并在大肠杆菌BL21(DE3)中诱导表达.以纯化的重组蛋白(rCpserpin)为包被抗原,优化反应条件,建立检测兔豆状囊尾蚴病血清抗体的间接ELISA方法.结果表明,Cpserpin基因的完整ORF长为1 149 bp,含有serpin家族保守基序(DEEGAE)、serpin标签序列(FKVDHPFIFFI)以及serpin特有的反应中心环结构域(RCL);rCpserpin主要以包涵体形式表达,且能被家兔豆状囊尾蚴阳性血清所识别.所建立的间接ELISA的最佳反应条件包括:rCpserpin包被质量浓度为2μg/mL,待检血清和酶标二抗的稀释度分别为1:100和1:2 500,包被液和封闭液分别为pH9.6碳酸盐缓冲液和10 g/L牛血清白蛋白.利用上述条件检测60份家兔豆状囊尾蚴阳性血清和108份阴性血清,证实该方法的临界值为0.368,敏感性和特异性分别为96.7％和93.5％.结果表明,基于Cpserpin重组蛋白的间接ELISA方法可用于家兔豆状囊尾蚴病的血清学诊断.

To identify and express the serine protease inhibitor of Taenia soliam(TsSerpin),the total RNA was extracted from T.solium metacestodes.Specific primers were designed and used to amplify TsSerpin4848 by RT-PCR.PCR products were ligated into the pMD19-T vector.After being transformed into Escherichia coli DH5a,the positive clones were identified and confirmed by sequencing.The sequence of TsSerpin4848 was analyzed with the bioinformatics software.The recombinant pET-30a(+)-4848 plasmid was constructed and transformed into E.coli BL21(DE3).The target protein was expressed,purified and analyzed by SDS-PAGE and Western-blot.In result,TsSerpin4848 cDNA sequence contained an open reading frame of 1 065 nucleotides,and the deduced protein consisted of 355amino acids with the theoretical protein molecular weight of 39 ku.TsSerpin4848 had a serpin conserved structure domain with the DEEGAE and FIVDHPFLFFI,and a specific reactive centre loop (RCL) motif.Analysis of tertiary structure of TsSerpin-4848 revealed that it had eight α-helices and three β-sheets.The expressed products of TsSerpin4848,existed in inclusion bodies,could be recognized by the positive serum from the pig infected with T.solium by immunoblotting,indicating that the protein TsSerpin4848 has a good immunogenicity and it is hopeful to be a diagnostic antigen for cysticercosis.

AbstractSerine protease inhibitors (serpins) play essential physiological roles in a wide range of biological processes. Serpins are researched limited inTaenia solium, although some are considered to participate in host immune responses. Tsserpins were identified as typical serpins due to the primary structure of characteristic features: the serpin motif, serpin signature and reaction centre loop (RCL). RCLs of four serpin genes (TsB6, Ts4848, Ts12383 and Ts570) contained the conserved sequences of inhibitory serpins, which may involve in immune regulation. TsEP45 differed greatly from the patterns of representative serpins, suggesting that TsEP45 may be non-inhibitory. The bioinformatic analyses were supposed that Tsserpins might be a potential antigen for diagnosis. The five recombinant Tsserpin proteins were expressed and identified reacting withCysticercus cellulosae-positive serum samples. The indirect enzyme-linked immunosorbent assay (iELISAs) based on Tsserpins were developed and validated, one of the five Tsserpins, TsEP45, showed excellent diagnostic results with 93·33% sensitivity and 94·12% specificity, respectively. This performance was in perfect accordance with the results of the bioinformatic analysis. This study provided a comprehensive demonstration of sequences and structural-based analysis of Tsserpins. The iELISAs based on five Tsserpins were developed and compared.TsEP45 was the potential species-specific antigen for developing iELISA to detect porcine cysticercosis.

Cysticercosis, caused by metacestodes of Taenia solium, has a significant soci-economic impact and is of considerable importance in public health. However, there are no specific diagnostic antigens to distinguish between T. solim and Taenia hydatigena. In the present study, cAMP-dependent protein kinase regulatory subunit (TsPKA-r), an excretory/secretary (ES) antigen of T. solium, was used to establish a specific and sensitive diagnostic tool for detection of porcine cysticercosis. The full-length sequence encoding TsPKA-r was amplified by PCR, sequenced and then identified by bioinformatics. The fusion protein with 6×His-tags was expressed in E. coli, purified by Ni Sepharose™ 6 Fast Flow and used to test reactionogenicity by immunoblotting. TsPKA-r based indirect enzyme-linked immunosorbent assays (iELISA) showed good performance in recognition of sera of pigs experimentally infected with T. solium metacestodes, with 93.88% sensitivity and 96.40% specificity. There were no cross-reactions against the sera from pigs experimentally infected with T. hydatigena, Toxoplasma gondii or Trichinella spiralis. These results indicate that the TsPKA-r is a promising immunodiagnostic antigen for detection of porcine cysticercosis.