关键词: 鸡, LTBP1, 原核表达, 单克隆抗体, 间接免疫荧光试验, 蛋白免疫印迹试验

Abstract: In order to study the function of latent transforming growth factor binding protein 1(LTBP1),the recombinant eukaryotic expression plasmid,pET-30a-LTBP1,was constructed and transformed into E.coli BL21 competent cells for the IPTG-induced protein expression. The recombinant LTBP1 protein was purified by denaturation and affinity chromatography,and used to immunize Balb/C mice.Cell fusion and hybridoma technology were performed to produce hybridomas and develop the monoclonal antibodies(mAbs) against chicken LTBP1 protein. The supernatant of specific antibodies were screened by indirect enzyme-linked immunosorbent assay(iELISA),and two hybridoma cell lines named as 3C11-A9 and 2B1-G7 steadily secreting antibodies against LTBP1 protein of chicken were obtained.The cell line 3C11-A9 with higher titers was chose for the preparation and purification of mAb in mouse ascites. Both of the indirect immunofluorescence assay(IFA) and Western blot analysis indicated that the 3C11-A9 mAb specifically reacted to chicken LTBP1 protein,and the IFA also demonstrated that 3C11-A9 mAb specifically stained the LTBP1 proteins expressed in eight cell lines derived from mammalians,including human being,monkey,pig and mouse.These data indicate that the chicken LTBP1-specific mAbs have been successfully developed,with a wide usage for the LTBP1.

Key words: Chicken, LTBP1, Eukaryotic expression, Monoclonal antibody, Indirect immunofluorescence assay, Western blot