捻转血矛线虫NADH:泛醌氧化还原酶结构域包含蛋白基因的克隆表达及功能分析

Cloning,Expression and Function Analysis of NADH:Ubiquinone Oxidoreductase Domain Containing Protein in Haemonchus contortus

选取捻转血矛线虫NADH:泛醌氧化还原酶结构域包含蛋白(NDUDC)基因进行克隆表达,并对其功能进行研究。采用RT-PCR方法对NDUDC基因进行克隆,构建表达载体得到重组蛋白质,Western blot分析蛋白质的抗原特性;采血分离山羊外周血单个核细胞(PBMCs)与重组蛋白质共培养,利用间接免疫荧光试验检验重组蛋白质与细胞的结合情况;以不同质量浓度(0、10、20、40μg·mL-1)重组蛋白质刺激细胞,测定其对细胞增殖、细胞迁移和NO分泌的影响。结果表明,重组蛋白质能够有效刺激细胞增殖,显著提高细胞迁移能力以及增加NO的分泌。可见NDUDC蛋白具有较强的抗原性,可刺激细胞发挥免疫功能。

The gene of NADH：ubiquinone oxidoreductase domain containing protein（NDUDC）of Haemonchus contortus was selected to clone,express,and the functions of this protein were studied in this paper.The gene of NDUDC was cloned by RT-PCR,then expression vector was constructed to obtain the recombinant protein.The antigenicity of the protein was analyzed by Western blot.Peripheral blood mononuclear cells（PBMCs）were separated from goat and cultured in vitro with the recombinant NDUDC to check the binding of the protein to PBMCs by indirect immunofluorescence assay.Different concentrations（0,10,20,40μg·mL-1）of the recombinant protein were used to stimulate PBMCs,and the effects of cell proliferation,cell migration and NO secretion were tested.The results showed that the recombinant protein NDUDC could stimulate cell proliferation effectively,also significantly enhance cell migration and increase the secretion of NO.These results indicate that NDUDC protein has strong antigenicity,and can stimulate cells to exert immune function.