人肥大细胞类糜蛋白酶基因的原核表达及其多克隆抗体的制备与鉴定

目的:克隆人肥大细胞类糜蛋白酶cDNA,并进行原核表达,制备重组蛋白免疫家兔,制备兔抗类糜蛋白酶多克隆抗体。方法:用RT-PCR技术克隆类糜蛋白酶cDNA,用Gateway技术构建表达载体,以大肠杆菌为宿主,用L-阿拉伯糖诱导重组肥大细胞类糜蛋白酶的表达,经Ni-NTA-Agarose柱层析纯化后,以其为免疫原制备兔抗类糜蛋白酶多抗,并以间接ELISA测定抗体效价,以Westernblot鉴定抗体的特异性。结果:成功地克隆了人肥大细胞类糜蛋白酶cDNA,并在大肠杆菌BL21-AI中表达成功,用纯化的重组类糜蛋白酶为免疫原,免疫家兔制备了兔抗类糜蛋白酶抗体,ELISA结果显示效价达1:12800,Western blot分析表明该抗体能特异结合类糜蛋白酶。结论:以纯化的重组类糜蛋白酶为免疫原,成功地制备了效价及特异性较高的兔抗类糜蛋白酶抗体,为进一步建立简便的类糜蛋白酶检测方法及研究类糜蛋白酶生物学功能奠定了良好的基础。

Prokaryotic expression of human mast cell chymase gene and preparation of its polyclonal antibody

AIM:To express the human mast cell chymase cDNA in E.coli and prepare the antibody against human mast cell chymase with recombinant chymase.METHODS:The human mast cell chymase cDNA was cloned by RT-PCR.The recombinant chymase was expressed in E.coli with L-Arabinose induction and purified by Ni-NTA agarose column.Then the purified chymase was used as immunogen to immunize the rabbit.The titer and specificity of the anti-chymase antibody from the rabbit were analyzed by indirect ELISA and Western blot,respectively.RESULTS:The recombinant chymase was successfully expressed in E.coli,and the polyclonal anit-chymase antibody was prepared by immunizing the rabbit with the purified recombinant chymase.The titer of the generated antiserum was detected to be 1:12 800 by ELISA.Western blot analysis showed this antiboday bound specifically with chymase.CONCLUSION:The anti-chymase antibody from the rabbit with high titer and specificity has been prepared with purified recombinant chymase as immunogen,which lays a foundation for further research into detection and function of chymase.