海兰白鸡CDCD14基因的克隆、表达及多克隆抗体的制备

Cloning,Prokaryotic Expression of CD14 Gene and Preparation of Polyclonal Antibody of Hy-line White Chicken

通过克隆获得海兰白鸡CD14基因全长,其与红原鸡CD14氨基酸同源性为98.9%,将其插入pET-30a表达载体中,原核表达获得重组蛋白,约为55 ku,纯化后免疫兔制备多克隆抗体。间接ELISA结果显示多克隆抗体效价为25 600,Western-blot及间接免疫荧光试验结果显示多克隆抗体能够与chCD14重组蛋白、BHK-21细胞内chCD14蛋白及鸡外周血液淋巴细胞内chCD14蛋白特异性结合。本试验表明:制备的多克隆抗体具有天然活性,为进一步研究chCD14在雏鸡组织当中的表达分布奠定基础。

The whole cluster of differentiation 14 （CD14） gene of Hy-line white chicken was cloned and compared with red Jungle fowl,and the amino acid homology was 98.9%,then Hy-line white chicken＇s whole CD14 gene was subeloned into prokaryotic expression vector pET-30a. The recombinant protein with molecular weight of 55 ku was induced in prokaryotie expression systerm. Then the purified recombinant protein was used as antigen for immunization of rabbit to prepare polyclonal antibody. The results of indirect ELISA showed that the titer of polyelonal antibody was 25 600. Furthermore, Western blot and indirect immunoinfluseent assay demonstrated that this polyelonal antibody could react with recombinant ehCD14 protein, BHK-21 cells transfected with PVAX-chCD14 plasmid and natural ehCD14 protein in chicken peripheral blood lymphocyte. It＇s concluded that the polyelonal antibody had natural activity and laid a foundation for espression area research of chCD14 protein in chickling tissue.