兔抗破骨细胞蛋白质酪氨酸磷酸酶多克隆抗体的制备和应用

doi:10.13481/j.1671-587x.20180339

吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (03): 661-666.doi: 10.13481/j.1671-587x.20180339

兔抗破骨细胞蛋白质酪氨酸磷酸酶多克隆抗体的制备和应用

刘少伟^1,2, 姜欢¹, 王晓龙³, 李梦红¹, 陈玉¹, 唐中元¹, 胡敏¹

1. 吉林大学口腔医院正畸科, 吉林长春 130021;
2. 吉林省牙发育及颌骨重塑与再生重点实验室, 吉林长春 130021;
3. 吉林大学口腔医院老年病科, 吉林长春 130021

收稿日期:2017-09-08 出版日期:2018-05-28 发布日期:2018-05-31
通讯作者: 胡敏,教授,博士研究生导师(Tel:0431-88796023,E-mail:humin@jlu.edu.cn) E-mail:humin@jlu.edu.cn
作者简介:刘少伟(1990-),男,河南省郑州市人,在读医学硕士,主要从事正畸患者牙根吸收方面的研究。
基金资助:
国家自然科学基金资助课题（81470764，81600898）；吉林省卫生厅科研基金资助课题（2014Z073）；吉林省教育厅科研项目资助课题（吉教科合字[2015]第531号）；吉林大学白求恩计划项目资助课题（2015406）

Preparation and application of rabbit anti-osteoclastic protein tyrosine phosphatase polyclonal antibody

LIU Shaowei^1,2, JIANG Huan¹, WANG Xiaolong³, LI Menghong¹, CHEN Yu¹, TANG Zhongyuan¹, HU Min¹

1. Department of Orthodontics, Stomatology Hospital, Jilin University, Changchun 130021, China;
2. Jilin Provincial Key Laboratory of Tooth Development and Jaw Bone Remodeling, Changchun 130021, China;
3. Department of Geriatric, Stomatology Hospital, Jilin University, Changchun 130021, China

Received:2017-09-08 Online:2018-05-28 Published:2018-05-31

摘要/Abstract

摘要： 目的：采用已构建的pET28a-ΔPTP-oc重组质粒纯化重组蛋白，制备兔抗破骨细胞蛋白质酪氨酸磷酸酶（PTP-oc）多克隆抗体并鉴定其性能。方法：将构建的pET28a-ΔPTP-oc重组质粒转化至BL21（DE3）中，应用异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组蛋白表达。表达产物经过Q柱阴阳离子交换和Ni柱亲和层析获得纯化的PTP-oc重组蛋白，将纯化的重组蛋白作为抗原免疫家兔，获得PTP-oc多克隆抗体。采用间接ELISA法检测抗体效价，Western blotting法检测抗体的特异性。结果：成功纯化出PTP-oc重组蛋白。间接ELISA法检测，采用纯化诱导后的重组蛋白免疫家兔获得了兔抗PTP-oc多克隆抗体，多克隆抗体效价达1∶32000以上，Western blotting法检测，所得多克隆抗体有较高的特异性。结论：成功纯化了PTP-oc重组蛋白，制备出PTP-oc多克隆抗体。

关键词: 效价, 破骨细胞蛋白质酪氨酸磷酸酶, 多克隆抗体, 破骨细胞, 鉴定

Abstract: Objective: To purify the recombinant protein by using the constructed pET28a-ΔPTP-oc recombinant expression plasmid,and to prepare the rabbit polyclonal antibody against osteoclastic protein tyrosine phosphatase(PTP-oc) and to identify its properties. Methods: The constructed pET28a-ΔPTP-oc recombinant plasmid was transformed into E.coli BL21(DE3),and the isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of recombinant protein.The recombinant protein was purified by the method of Q fast flow agarose and Ni-NTA agarose,and the purified recombinant protein was used as an antigen to immunize the rabbit to obtain PTP-oc polyclonal antibody.The titer and specificity of the antibody were identified by indirect ELISA and Western blotting methods. Results: The recombinant PTP-oc protein was successfully purified.The indirect ELISA results showed that after immunizing the rabbit with the recombinant PTP-oc protein,the polyclonal antibody with the titer of 1:3 2000 was gotten.The Western blotting results showed that the polyclonal antibody had high specificity. Conclusion: The PTP-oc recombinant protein is successfully purified,and the polyclonal antibody of PTP-oc is prepared successfully.

Key words: titer, osteoclastic protein-tyrosine phosphatase, polyclonal antibody, osteoclast, identification

中图分类号:

Q814

刘少伟, 姜欢, 王晓龙, 李梦红, 陈玉, 唐中元, 胡敏. 兔抗破骨细胞蛋白质酪氨酸磷酸酶多克隆抗体的制备和应用[J]. 吉林大学学报(医学版), 2018, 44(03): 661-666.

LIU Shaowei, JIANG Huan, WANG Xiaolong, LI Menghong, CHEN Yu, TANG Zhongyuan, HU Min. Preparation and application of rabbit anti-osteoclastic protein tyrosine phosphatase polyclonal antibody[J]. Journal of Jilin University Medicine Edition, 2018, 44(03): 661-666.

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兔抗破骨细胞蛋白质酪氨酸磷酸酶多克隆抗体的制备和应用

Preparation and application of rabbit anti-osteoclastic protein tyrosine phosphatase polyclonal antibody

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