Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.
Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.
复制标题
通过cDNA表达文库筛选鉴定巨型艾美耳球虫免疫保护基因
DOI：
10.1186/s13071-017-2029-4
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发表时间：
2017-02-16
影响因子：
3.2
通讯作者：
Song X
中科院分区：
文献类型：
--
作者：
Yang X;Li M;Liu J;Ji Y;Li X;Xu L;Yan R;Song X
背景极大艾美耳球虫是引起禽球虫病的最常见的球虫之一，给全球养殖业造成巨大的经济损失。目前的控制策略，如抗球虫药物和活疫苗，由于其缺点而受到限制。包括重组疫苗和DNA疫苗在内的第三代抗球虫疫苗被认为是一种很有前途的替代策略。到目前为止，极大棘球绦虫保护性抗原的报道还很少。因此，迫切需要寻找新的巨型埃希氏菌保护性抗原，为新型抗球虫疫苗的研制奠定基础。方法利用Gateway技术构建极大埃希氏菌新的保护性基因文库。随后，将表达文库划分为15个亚文库，用于鸡寄生虫攻击模型的cDELI。结果采用Gateway技术构建了一个高质量的入库文库，包含9.2个 × 106克隆，平均插入片段长度为1.63kb。表达文库容量为2.32 × 107个集落形成单位，平均插入片段长度为1.64kb。用鸡寄生虫攻击模型筛选表达文库。筛选得到6个免疫保护基因，包括EmJS-1、EMRP、EmHP-1和EmHP-2四个新的保护基因，以及两个已知的保护基因EmSAG和EmCKRS。EmJS-1是极大拟青霉的selR结构域蛋白，其功能未知。EmHP-1和EmHP-2是极大乳杆菌的假想蛋白。EMRP和EmSAG分别是巨型棘球绦虫的菱形蛋白和表面抗原糖蛋白，参与了该寄生虫的侵袭。结论为进一步筛选极大棘球绦虫T细胞刺激或抑制抗原提供了一个表达文库。此外，我们的结果还提供了六种候选保护性抗原，可用于开发新的巨型埃希氏菌疫苗。
BackgroundEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.MethodsWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.ResultsAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.ConclusionsOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.