豆状带绦虫Tp18基因的原核表达及其产物的抗原性分析

Prokaryotic Expression of Tp18Gene fromTaenia pisiformis and Antigenicity Analysis of the Expressed Product

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14d),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32ku,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DotELISA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。

In order to evaluate the effect of immunoprotection and diagnosis against rabbit Taenia pisiforrnis cysticercosis by recombinant Tpl8 （rTpl8） 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 rTpl8 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 rTpl8 protein group and PBS group was car- ried 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. pisi formis from rabbit by Western blotting. Vaccination trials showed that the rTp18 protein induced effective immune protection against T. pisiformis cysticercosis with 95.59O/oo reduction （97.38~/oo 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 specifici- ty of the dot-ELISA were 90.74% （49/54） and 96.52% （111/115）, respectively. These promis- ing results indicate that rTpl8 protein is suitable for the development of effective vaccination and diagnositic strategies against cysticercosis in rabbits, providing a foundation for control of T. pisi f ormis cysticercosis.