Galectin-7多克隆抗体的制备与鉴定

Preparation and Characterization of Polycolnal Antibody of Galectin-7

目的：制备鼠抗重组半乳糖凝集素7（Galectin-7）多克隆抗体及其在膀胱癌组织中特异性鉴定。方法PCR 法从人包皮组织中扩增 Galectin-7基因，并构建到 pET28a 表达载体中，使用 IPTG 诱导重组蛋白的表达，通过镍柱亲和层析纯化获得 Galectin-7蛋白。以纯化的 Galectin-7蛋白混合福氏完全佐剂免疫 Balb／C 小鼠制备抗体，并用 ELISA，Western blot 及免疫组织化学法检测抗体。结果成功表达、纯化了 Galectin-7重组蛋白，ELISA 检测 Galectin-7蛋白免疫的小鼠血清效价为1∶32000，Western blot 显示该抗体能与天然的 Galectin-7特异结合，免疫组织化学法检测结果表明该抗体识别人膀胱肿瘤组织的天然 Galectin-7。结论通过制备重组 Galectin-7蛋白为免疫原，免疫家兔，成功地制备了效价高、特异性好的抗 Galectin-7多克隆抗体。

Objective To prepare the mouse anti recombinant human Galectin-7 antibody and the antibody was characterized in bladder cancer.Methods The gene coding for Galectin-7 was amplified by PCR from the cDNA of human foreskin cells and cloned into prokaryotic expression vector pET28a.Then the recombinant plasmid pET28a/Galectin-7 was transformed into E.coli BL21 （DE3）and expressed under IPTG induction.The recombinant Galectin-7 was purified through Ni2＋-NT agarosegel column and the purified Galectin-7 used as imunogen to imunize the mouse.The titer and specificity of the anti-Galectin-7antibody from the mouse were analyzed by ELISA,Western blot and immunohistochemistry,respectively.Results The recombinant Galectin-7 was successfully expressed and purified,and the polyclonal ani-Galectin-7 antibody was suc-cessfully prepared.The titer of the antiserum was 1∶32 000 by ELISA.Western blot analysis showed this antiboday reacted specifically with Galectin-7.Immunohistochemistry analysis showed the antibody could recognize the native Galectin-7 in the human bladder cancer tissue.Conclusion The preparation recombinant Galectin-7 protein was as immunogen in rabbits.It was successful to produce high titer and high specificity of anti Galectin-7 polycolonal antibody.