宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响

Effects of Host Glyceraldehyde-3-phosphate Dehydrogenase on Eimeria tenella Sporozoites Invasion of Cells

目前,关于宿主细胞应对球虫感染机制的研究极其有限。在前期研究中,我们利用相对和绝对定量同位素标记技术结合液相色谱串联质谱技术分析了感染柔嫩艾美耳球虫子孢子72 h后鸡胚成纤维(CEF)细胞的蛋白质组表达变化,作者拟进一步研究其中一个上调蛋白——甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)对柔嫩艾美耳球虫子孢子入侵细胞的影响。克隆鸡GAPDH基因并构建原核重组质粒pGEX-4T-1-GAPDH,表达重组GAPDH蛋白,用Western blot对表达进行验证。使用q-PCR、Western blot和免疫组织化学的方法检测感染柔嫩艾美耳球虫子孢子后DF-1细胞中GAPDH的表达量,并使用抗体抑制试验检测GAPDH多克隆抗体对柔嫩艾美耳球虫子孢子入侵细胞的影响。结果显示:成功克隆1 123 bp的GAPDH基因,并获得61.7 ku的重组GAPDH蛋白。q-PCR和免疫组织化学检测均显示,GAPDH在柔嫩艾美耳球虫子孢子感染的DF-1细胞中的表达显著升高。抗体抑制试验表明,与相同浓度的正常兔IgG相比,50、100、200、300和400μg·mL-1的抗GAPDH抗体均能明显抑制子孢子的入侵。以上结果表明,子孢子感染会引起宿主细胞GAPDH蛋白的上调,并且宿主GAPDH蛋白在子孢子入侵过程中起正调控作用。

At present,the mechanism of how the host cell responds to Eimeria tenella infection is highly limited.In our previous study,isobaric tags for relative and absolute quantitation were used to study the proteomics changes in host cells after infection with E.tenella sporozoites.In this study,one of the up-regulated proteins,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),was selected to study its effect on the invasion of Eimeria tenella sporozoites.Gallus gallus GAPDH gene was cloned,and the prokaryotic recombinant expression plasmid pGEX-4T-1-GAPDH was constructed.The recombinant GAPDH protein(rGAPDH)was expressed and identified by Western blot.Expression of GAPDH in DF-1 cells during E.tenella sporozoites infection was detected by q-PCR,Western blot and immunohistochemistry.The effect of GAPDH polyclonal antibody on sporozoites invasion of cells was detected by antibody inhibition assay.Results were as follows:A 1 123-bp GAPDH was cloned and the rGAPDH was recognized as a^61.7 kDa protein by Western blot.The expression of GAPDH in E.tenella sporozoites-infected DF-1 cells were up-regulated by q-PCR and immunohistochemistry detection.The antibody inhibition assay showed that antibodies against GAPDH at 50,100,200,300 and 400μg·mL^-1 could significantly inhibit sporozoite invasion compared with the same does of normal rabbit IgG.These results show that E.tenella sporozoites infection could induce the up-regulation of host GAPDH,and host GAPDH plays an active role in Eimeria invasion.