地克珠利对柔嫩艾美耳球虫丝氨酸/苏氨酸磷酸酶5表达的影响

Effect of diclazuril on serine/threonine protein phosphatase type 5 expression in Eimeria tenella

建立地克珠利抗柔嫩艾美耳球虫(Eimeria tenella)感染鸡动物模型,收集获得E. tenella第2代裂殖子,通过PCR扩增E. tenella第2代裂殖子丝氨酸/苏氨酸磷酸酶5(serine/threonine protein phosphatase type 5,Et PP5)基因编码序列,构建pET-28a-Et PP5表达质粒,转化至BL21(DE3)感受态细胞,经IPTG诱导表达融合蛋白,镍亲和层析柱法纯化表达蛋白并免疫BABL/c小鼠制备Et PP5抗血清。采用免疫荧光和Western-blot技术检测第2代裂殖子Et PP5蛋白的表达。结果显示,30℃时,用1 mmol/L的IPTG诱导pET-28a-Et PP5表达的融合蛋白大多以包涵体形式存在。Et PP5主要分布在E. tenella第2代裂殖子的细胞质中,细胞核也有少量分布。经地克珠利处理后,E. tenella第2代裂殖子Et PP5蛋白表达降低了47.65%(P<0.01)。提示,地克珠利可能通过作用Et PP5发挥抗球虫作用,这为地克珠利抗球虫作用机理的阐述提供了理论依据。

The animal model of diclazuril against Eimeria tenella(E.tenella)infection in chickens was established and the second-generation merozoites of E.tenella were prepared for the study.The coding sequence of serine/threonine protein phosphatase type 5 gene of E.tenella(EtPP5)was amplified via PCR.The expression plasmid pET-28a-EtPP5 was constructed and transformed into Escherichia coli BL21(DE3)competent cells.The fusion protein induced expression by IPTG was purified via nickel affinity chromatography.Anti-EtPP5 polyclonal antibody was prepared by immunizing BABL/c mice.The EtPP5 protein expression was detected by immunofluorescence analysis and Western blot technology,respectively.Results showed that most of the fusion proteins expressed by pET-28a-EtPP5 were in the form ofinclusion bodies under induction of 1 mmol/L IPTG at 30℃.EtPP5 was mainly distributed in cytoplasm and a small amount in nucleus.The diclazuril treatment significantly decreased the EtPP5 protein expression in the second-generation merozoites of E.tenella.It is suggested that diclazuril may exerts anticoccidial effect by acting on EtPP5 protein,which provides a theoretical basis for the elaboration of the anticoccidial action mechanism of diclazuril.