豆状带绦虫Tp18基因的原核表达及其产物的抗原性分析

doi:10.11843/j.issn.0366-6964.2013.11.017

摘要/Abstract

摘要：

为评价豆状带绦虫Tp18重组蛋白对兔豆状囊尾蚴病的免疫保护效果和诊断应用价值，从豆状带绦虫成虫转录组数据库中筛选到Tp18基因，并进行克隆和原核表达，以纯化后的重组蛋白作为免疫原和反应原。免疫保护试验设计了重组蛋白组、佐剂组和PBS组，共免疫2次（间隔14 d），二免后的第7天用5 000个豆状带绦虫虫卵攻虫；攻虫后的第49天屠宰实验兔，统计兔体内豆状囊尾蚴的数量。整个试验过程中每周采血，分离血清进行IgG和IgA水平检测。同时，设计了Tp18重组蛋白和PBS重复组，试验程序和检测指标均与第一次试验相同。此外，采用Tp18重组蛋白建立了诊断家兔豆状囊尾蚴病的Dot-ELISA方法，对169份家兔血清进行分析，评估其诊断应用价值。结果表明表达的Tp18基因成熟肽的重组蛋白约为32 ku， Western blotting证明该重组蛋白与豆状带绦虫感染兔阳性血清具有良好的反应原性。Tp18重组蛋白组的减虫率为95.59%（重复组为97.38%），极显著高于佐剂组和PBS组（P<0.01）；诱导产生的特异性抗体为IgG。以病原学检查为金标准，Tp18重组蛋白建立的Dot-ELISA诊断方法具有较高的敏感性（49/54，90.74%）和特异性（111/115，96.52%）。研究结果表明，Tp18重组蛋白可作为豆状带绦虫的疫苗抗原和诊断抗原，为兔豆状囊尾蚴病的防控提供了理论基础。

Abstract:

In order to evaluate the effect of immunoprotection and diagnosis against rabbit Taenia pisiformis cysticercosis by recombinant Tp18 (rTp18) protein, Tp18 gene was screened from transcriptome of adult T. pisiformis, then was cloned and expressed. rTp18 protein was used as immunogen and atopen. The samples were grouped into three experimental groups, including rTp18 protein group, PBS group and adjuvant group. Immunization was performed twice at 14-day intervals. Seven days after the second immunization, each rabbit was experimentally infected orally with 5 000 mature viable T. pisiformis eggs, and sacrificed at 49 days post-infection. The number of cysticercoids was counted. By the end of the experiment, serum was collected for detection of IgG and IgA every week. Repeated trial of rTp18 protein group and PBS group was carried out using identical procedures and conditions. The molecular weight of rTp18 protein is about 32 kDa. rTp18 protein was proved to be well reacted with the positive sera against T. pisiformis from rabbit by Western blotting. Vaccination trials showed that the rTp18 protein induced effective immune protection against T. pisiformis cysticercosis with 95.59% reduction （97.38% reduction in repeated trial） in metacestode burdens of vaccinated rabbits, which was significantly higher than PBS and adjuvant group (P<0.01). IgG was the antibody of humoral immunity. Meanwhile, dot-ELISA was used to diagnose T. pisiformis cysticercosis for rabbits. Based on the results of rabbit necropsy of 169 abattoir rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 90.74% (49/54) and 96.52% (111/115), respectively. These promising results indicate that rTp18 protein is suitable for the development of effective vaccination and diagnositic strategies against cysticercosis in rabbits, providing a foundation for control of T. pisiformis cysticercosis.

中图分类号:

S852.734

杨德英，陈林，古小彬，赖松家，孙家刚，杨光友. 豆状带绦虫Tp18基因的原核表达及其产物的抗原性分析[J]. 畜牧兽医学报, 2013, 44(11): 1819-1825.

YANG De-ying, CHEN Lin, GU Xiao-bin, LAI Song-jia, SUN Jia-gang,YANG Guang-you. Prokaryotic Expression of Tp18 Gene from Taenia pisiformis and Antigenicity Analysis of the Expressed Product[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1819-1825.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2013.11.017

https://www.xmsyxb.com/CN/Y2013/V44/I11/1819

参考文献

［1］BETANCOURT M A, DE ALUJA A S, SCIUTTO E, et al. Effective protection induced by three different versions of the porcine S3Pvac anticysticercosis vaccine against rabbit experimental Taenia pisiformis cysticercosis［J］. Vaccine, 2012, 30(17): 2760-2767.

［2］SCHANTZ P M. Progress in diagnosis, treatment and elimination of echinococcosis and cysticercosis［J］. Parasitol Int, 2006, 55(Suppl): S7-S13.

［3］周永学, 杜爱芳, 张雪娟, 等. 肉兔豆状囊尾蚴病的危害性研究［J］. 浙江农业科学, 2008, 3: 372-373.

［4］LIGHTOWLERS M W, COLEBROOK A L, GAUCI C G, et al. Vaccination against cestode parasites: anti-helminth vaccines that work and why［J］. Vet Parasitol, 2003, 115(2): 83-123.

［5］TARIGAN S. Protective value of immune responses developed in goats vaccinated with insoluble proteins from Sarcoptes scabiei［J］. JITV, 2005, 10(2): 118-126.

［6］RASSY D, BOBES R J, ROSAS G, et al. Characterization of S3Pvac anti-cysticercosis vaccine components: implications for the development of an anti-cestodiasis vaccine［J］. PLoS One, 2010, 5(6): e11287.

［7］JABBAR A, VERASTEGUI M, LACKENBY J A, et al. Variation in the cellular localization of host-protective oncospheral antigens in Taenia saginata and Taenia solium［J］. Parasite Immunol, 2010, 32(9-10): 684-695.

［8］CAI X, YUAN G, ZHENG Y, et al. Effective production and purification of the glycosylated TSOL 18 antigen，which is protective against pig cysticercosis［J］. Infect Immun, 2008, 76(2): 767-770.

［9］GAUCI C, VURAL G, ÖNCEL T, et al. Vaccination with recombinant oncosphere antigens reduces the susceptibility of sheep to infection with Taenia multiceps［J］. Int J Parasitol, 2008, 38(8-9):1041-1050.

［10］ASSANA E, GAUCI C G, KYNGDON C T, et al. Antibody responses to the host-protective Taenia solium oncosphere protein TSOL18 in pigs are directed against conformational epitopes［J］. Parasite Immunol, 2010, 32(6): 399-405.

［11］JIANG L, XU X N, LI X, et al. Identification of the immunodominant regions of the Em18 antigen and improved serodiagnostic specificity for alveolar echinococcosis［J］. Parasite Immunol, 2004, 26(10):377-385.

［12］刘红霞, 才学鹏, 张少华, 等. 重组 CE18 蛋白在绵羊绦虫蚴病诊断上的应用［J］. 中国农业科学，2009, 42(8): 2997-3002.

［13］SAKO Y, NAKAO M, NAKAYA K, et al. Alveolar echinococcosis: characterization of diagnostic antigen Em18 and serological evaluation of recombinant Em18［J］. J Clin Microbiol, 2002, 40(8):2760-2765.

［14］YANG D Y, FU Y, WU X H, et al. Annotation of the transcriptome from Taenia pisiformis and its comparative analysis with three Taeniidae species［J］. PLoS ONE, 2012, 7(4): e32283.

［15］BIAN Y, CHEN W, YANG G Y, et al. Cloning, expression and evaluation of the efficacy of a recombinant Haemaphysalis concinna Hc-23 antigen in rabbits［J］. Vaccine, 2011, 29(5):1041-1044.

［16］HU Y X, GUO J Y, SHEN L, et al. Get effective polyclonal antisera in one month［J］. Cell Res, 2002, 12(2): 157-160.

［17］PIÑA R, GUTIÉRREZ A H, GILMAN R H, et al. A dot-ELISA using a partially purified cathepsin-L-like protein fraction from Taenia solium cysticerci for the diagnosis of human neurocysticercosis［J］. Ann Trop Med Parasitol, 2011, 105(4): 311-318.

［18］VARGHESE A, RAINA O K, NAGAR G, et al. Development of cathepsin-L cysteine proteinase based dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes［J］. Vet Parasitol, 2012, 183(3-4): 382-385.

［19］CRAIG P S, ZUMBUEHL O. Immunization against experimental rabbit cysticercosis using liposome-associated antigen preparations［J］. J Helminthol, 1988, 62(1): 58-62.

［20］RAJASEKARIAH G R, RICKARD M D, O'DONNELL I J. Taenia pisiformis: protective immunization of rabbits with solubilized oncospheral antigens［J］. Exp Parasitol, 1985, 59(3): 321-327.

［21］周永学, 杜爱芳, 张雪娟, 等. 我国兔豆状囊尾蚴病的研究进展［J］. 中国养兔, 2006, 1: 25-28.

［22］单永利, 张宝庆, 王双同. 现代养兔技术［M］. 北京: 中国农业出版社, 2004.

［23］MIGUEL-ANGEL B A, AGUSIN O, VIRGINIO A, et al. Changes in behavioural and physiological parameters associated with Taenia pisiformis infection in rabbits (Oryctolagus cuniculus) that may improve early detection of sick rabbits［J］. Worl Rab Sci, 2011, 19(1): 21-30.

［24］CRAIG P S. Surface-associated proteins and host IgG on early and late metacestode stages of Taenia pisiformis［J］. Parasite Immunol, 1998, 10(3): 243-254.

［25］王晓霞, 骆学农, 孙晓林, 等. 豆状囊尾蚴人工感染家兔抗体水平的消长［J］. 中国兽医科学, 2009, 39(4): 283-286.

［26］CRAIG P S. Circulating antigens, antibodies and immune complexes in experimental Taenia pisiformis infections of rabbits［J］. Parasitology, 1984, 89:121-131.

［27］韩进欢, 张小宇, 张倩, 等. 豆状带绦虫不同发育阶段虫体的抗原特性分析［J］. 中国兽医科学, 2012, 42(12):1259-1263.

［28］FERRER E, BONAY P, FOSTER-CUEVAS M, et al. Molecular cloning and characterisation of Ts8B1, Ts8B2 and Ts8B3, three new members of the Taenia solium metacestode 8kDa diagnostic antigen family［J］. Mol Biochem Parasitol, 2007, 152(1):90-100.

［29］SWARNA S R, PARIJA S C. Dot-Elisa for evaluation of hydatid cyst wall, protoscoleces and hydatid cyst fluid antigens in the serodiagnosis of cystic echinococcosis［J］. Rev Inst Med trop S Paulo, 2008, 50(4): 233-236.

［30］PRASAD A, NASIR A, SINGH N. Detection of anti-Haemonchus contortus antibodies in sheep by dot-ELISA with immunoaffinity purified fraction of ES antigen during prepatency［J］. Indian J Exp Biol, 2008, 46(2): 94-99.

［31］SHARMA R, TUTEJA U, KHUSHIRAMANI R, et al. Application of rapid dot-ELISA for antibody detection of leptospirosis［J］. J Med Microbiol, 2007, 56(6): 873-874.

[1]	王正荣, 马勋, 张艳艳, 孙艳, 孟季蒙, 薄新文. 细粒棘球绦虫原头蚴、包囊壁和成虫circRNA差异表达分析[J]. 畜牧兽医学报, 2023, 54(8): 3474-3489.
[2]	杜小迪, 侯巍, 苏中华, 马青梅, 何雪, 华瑞其, 阳爱国, 杨光友. 细粒棘球绦虫泛素结合酶基因家族的生物信息学及表达分析[J]. 畜牧兽医学报, 2023, 54(6): 2605-2618.
[3]	李红, 李艳萍, 刘婷丽, 陈国梁, 王立群, 郭小腊, 骆学农. 多房棘球蚴感染对小鼠肝脂质代谢的影响[J]. 畜牧兽医学报, 2023, 54(1): 263-271.
[4]	李艳萍, 刘婷丽, 李红, 陈国梁, 王立群, 郭小腊, 骆学农. 多房棘球蚴诱导小鼠枯否氏细胞极化相关ceRNA调控网络的构建与分析[J]. 畜牧兽医学报, 2022, 53(12): 4410-4418.
[5]	王正荣, 马勋, 张艳艳, 孟季蒙, 薄新文. 基于转录组测序分析细粒棘球绦虫原头蚴与巨噬细胞RAW264.7免疫互作相关基因[J]. 畜牧兽医学报, 2022, 53(11): 3975-3988.
[6]	何雪, 王凝, 华瑞其, 杜小迪, 杨光友. 细粒棘球绦虫BAG3和EB1基因的克隆、表达及其在原头蚴细胞凋亡中的作用[J]. 畜牧兽医学报, 2022, 53(5): 1562-1575.
[7]	王正荣, 马勋, 张艳艳, 孟季蒙, 薄新文. Th1/Th2型免疫反应相关基因在巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激时的表达谱研究[J]. 畜牧兽医学报, 2022, 53(1): 250-262.
[8]	尚婧晔, 张光葭, 丹巴泽里, 王奇, 喻文杰, 何伟, 廖沙, 沈芸舟, 黄燕, 王谦, 钟波, 刘阳. 基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查[J]. 畜牧兽医学报, 2021, 52(12): 3546-3556.
[9]	潘耀谦, 孔令芸, 李鹏, 岳峰, 吴玉平, 刘兴友. 一种新型无钩豆状囊尾蚴的鉴定和18S DNA序列分析[J]. 畜牧兽医学报, 2019, 50(11): 2283-2289.
[10]	王正荣, 张艳艳, 马勋, 刘乙, 徐梦飞, 孟季蒙, 薄新文. 细粒棘球绦虫原头蚴lncRNA表达谱分析[J]. 畜牧兽医学报, 2019, 50(9): 1864-1873.
[11]	郭承, 万洁, 杨应东, 文建国, 刘俞辰, 古小彬, 谢跃, 杨光友. 脑多头蚴重组蛋白rTm16和rTm-GST的免疫保护效果分析[J]. 畜牧兽医学报, 2018, 49(12): 2707-2714.
[12]	王正荣, 薄新文, 张艳艳, 马勋, 卢平萍, 徐梦飞, 孟季蒙. 细粒棘球绦虫原头蚴microRNA表达谱分析[J]. 畜牧兽医学报, 2018, 49(11): 2477-2485.
[13]	徐梦飞, 卢平萍, 马勋, 张艳艳, 王炜烨, 孟季蒙, 王正荣, 薄新文. 细粒棘球绦虫蛋白质组学研究进展[J]. 畜牧兽医学报, 2018, 49(3): 466-476.
[14]	吴茂迪, 宋星桔, 闫敏, 阳爱国, 郭莉, 王凝, 谢跃, 古小彬, 杨光友. 细粒棘球绦虫半胱氨酸蛋白酶抑制剂基因的克隆、表达及诊断价值的初步评价[J]. 畜牧兽医学报, 2018, 49(3): 572-579.
[15]	黄兴, 徐璟, 王宇, 杨应东, 万洁, 郭承, 古小彬, 谢跃, 杨光友. 多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析[J]. 畜牧兽医学报, 2017, 48(12): 2374-2382.