地克珠利对柔嫩艾美耳球虫SAG4表达的影响

Effect of diclazuril on SAG4 expression in Eimeria tenella

建立地克珠利抗柔嫩艾美耳球虫感染鸡动物模型,收集获得柔嫩艾美耳球虫第2代裂殖子,采用PCR扩增柔嫩艾美耳球虫第2代裂殖子SAG4基因不含N端信号肽的编码序列,构建p ET-28a-SAG4表达质粒,转化至BL21(DE3)感受态细胞,用IPTG诱导表达融合蛋白,镍亲和层析柱法纯化蛋白,通过免疫BALB/c小鼠制备SAG4抗血清。用Real-time PCR检测SAG4 m RNA的表达,用Western-blot和免疫荧光技术检测SAG4蛋白的表达情况。结果显示,p ET-28a-SAG4表达的重组蛋白为可溶性蛋白,地克珠利显著降低了柔嫩艾美耳球虫第2代裂殖子SAG4基因m RNA和蛋白的表达。这提示SAG4基因可能与地克珠利抗球虫作用相关,这将为地克珠利抗球虫作用机理的阐述提供理论依据。

Diclazuril anticoccidiosis animal model was established and the second-generation merozoites of Eimeria tenella were prepared for further study.The ORF without a N terminal signal peptide of SAG4 gene in E.tenella was amplified and inserted into the expression vector pET-28a（＋）.The recombinant p ET-28a-SAG4 was transformed into Escherichia coli BL21（DE3）. The recombinant protein was induced to express by IPTG and identified by SDS-PAGE.SAG4 polyclonal antibodies were prepared by immunizing BALB/c mice.The mRNA ex pression of SAG4 was determined by real-time PCR,and protein expression of SAG4 was analyzed by Western-blot and immunofluorescence.In result,pET-28a-SAG4 was able to be expressed as a soluble protein.SAG4 antiserum was prepared from BALB/c mice immunized with the recombinant protein.The diclazuril treatment decreased the mRNA and protein expression of SAG4 of E.tenella.The results suggest that SAG4 may be involved in anticoccidiosis effect of diclazuril,which provide a theoretical basis for potential mechanism evaluation of diclazuril action on E. tenella.