Chen Zhang-Quan, Huang Zhen, He Tian-Wen, Lu Tian-Tian, Liang Xiao-Dong, He Shao-Heng
Department of Clinical Immunology, Guangdong Medical College, Zhanjiang 524023, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Nov;23(11):1025-7.
To express the human mast cell chymase cDNA in E.coli and prepare the antibody against human mast cell chymase with recombinant chymase.
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.
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 antibody bound specifically with chymase.
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.
在大肠杆菌中表达人肥大细胞糜蛋白酶cDNA，并利用重组糜蛋白酶制备抗人肥大细胞糜蛋白酶抗体。
通过逆转录聚合酶链反应（RT-PCR）克隆人肥大细胞糜蛋白酶cDNA。利用L-阿拉伯糖诱导在大肠杆菌中表达重组糜蛋白酶，并用镍-亚氨基二乙酸琼脂糖柱进行纯化。然后将纯化的糜蛋白酶用作免疫原免疫兔子。分别通过间接酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法分析兔抗糜蛋白酶抗体的效价和特异性。
重组糜蛋白酶在大肠杆菌中成功表达，用纯化的重组糜蛋白酶免疫兔子制备了多克隆抗糜蛋白酶抗体。通过ELISA检测所产生抗血清的效价为1:12 800。蛋白质免疫印迹分析表明该抗体与糜蛋白酶特异性结合。
以纯化的重组糜蛋白酶为免疫原制备了效价高且特异性强的兔抗糜蛋白酶抗体，为进一步研究糜蛋白酶的检测及功能奠定了基础。
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