猪细胞周期蛋白依赖性激酶2多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2015.08.006

›› 2015, Vol. 42 ›› Issue (8): 1956-1962.doi: 10.16431/j.cnki.1671-7236.2015.08.006

猪细胞周期蛋白依赖性激酶2多克隆抗体的制备与鉴定

李晓蓉, 唐青海, 卜宾, 毛倩倩, 李羽, 唐存多, 姚伦广, 焦铸锦, 阚云超

南阳师范学院, 南阳市兽医生物工程技术研究中心, 河南省伏牛山昆虫生物学重点实验室, 昆虫生物反应器河南省工程实验室, 南阳 473061

收稿日期:2015-01-23 出版日期:2015-08-20 发布日期:2015-08-27
通讯作者: 唐青海, 阚云超 E-mail:qinghaitang109@126.com;kanyunchao@163.com
作者简介:李晓蓉(1990-),女,河南信阳人,硕士生,研究方向:生物制药与疫苗工程,E-mail:1549857684@qq.com
基金资助:
国家自然基金青年基金(31101837);河南省重点科技攻关项目(142102110101);南阳师范学院引进人才专项项目(70640)

Preparation and Identification of Polyclonal Antibodies against Porcine Cyclin Dependent Kinase 2

LI Xiao-rong, TANG Qing-hai, BU Bin, MAO Qian-qian, LI Yu, TANG Cun-duo, YAO Lun-guang, JIAO Zhu-jin, KAN Yun-chao

Henan Provincial Engineering Laboratory of Insect Bio-reactor, Henan Provincial Key Laboratory of Insect Biology in Funiu Mountain, Center for Nanyang Veterinary Biological Engineering Technology, Nanyang Normal University, Nanyang 473061, China

Received:2015-01-23 Online:2015-08-20 Published:2015-08-27

摘要/Abstract

摘要： 为研究猪细胞周期蛋白依赖性激酶2(pCDK2)的生物学功能,本试验构建重组原核表达载体pET28a-pCdk2,转化大肠杆菌BL21 (DE3)受体菌,用IPTG诱导重组蛋白(rpCDK2)表达并进行SDS-PAGE和Western blotting鉴定,蛋白经纯化及与弗氏佐剂乳化后免疫家兔制备多克隆抗体。分别用Western blotting和免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性。结果显示,成功表达了rpCDK2蛋白,该蛋白以可溶性和包涵体两种形式存在,可溶性rpCDK2蛋白分子质量约为38 ku,包涵体rpCDK2蛋白在38~43 ku间有3种不同分子质量形式;IPMA检测结果显示,所制备的pCDK2蛋白多克隆抗体与猪肾细胞(PK-15)和猪睾丸细胞(ST)免疫染色呈特异性反应,且Western blotting分析显示,多克隆抗体与这两种细胞的总蛋白反应出现4条特异性条带(分子质量在34~50 ku之间),可能跟pCDK2的多种磷酸化形式有关。本试验利用原核表达系统成功制备了rpCDK2蛋白,并制备了免疫活性和特异性良好的pCDK2蛋白多克隆抗体,为该蛋白生物学功能及相关疾病研究提供了基础材料。

关键词: 猪细胞周期蛋白依赖性激酶2; 多克隆抗体; 鉴定

Abstract: In order to research the biofunction of porcine cyclin dependent kinase 2 (pCDK2), this study prepared polyclonal antibodies against pCDK2.The recombinant prokaryotic expression plasmid pET28a-pCdk2 was constructed and transformed into BL21 (DE3), IPTG was used to induce the expression of recombinant pCDK2 (rpCDK2) which was then analyzed by SDS-PAGE and Western blotting.The expressed proteins were purified and emulsified with freund's adjuvant, the mixture was used to immunize the rabbits to prepare polyclonal antibodies against pCDK2.The immunocompetence of the antibody was identified by immunoperoxidase monolayer assay (IPMA), and the specificity was confirmed by Western blotting.The results showed that rpCDK2 was successfully expressed by prokaryotic expression system, and the expression products showed two forms:Solubility and inclusion body(IB), the molecular weight of the solute rpCDK2 was about 38 ku, however three different molecular weight forms (from 38 to 43 ku) of IB were observed.IPMA results showed that polyclonal antibodies against pCDK2 protein showed perfect activity which could recognize pCDK2 expressed in PK-15 cells and ST cells specifically.Western blotting analysis showed that four bands with different molecular weight forms (from 34 to 50 ku) were observed when the polyclonal antibodies against pCDK2 protein reacted with the total protein extracted from PK-15 and ST cells.In conclusion, the rpCDK2 protein was successfully expressed by prokaryotic expression system, the prepared rabbit polyclonal antibodies against pCDK2 protein showed wonderful activity and specificity, and the study provided basic material for the research of protein biofunction and related diseases.

Key words: porcine cyclin dependent kinase 2; polyclonal antibody; identification

中图分类号:

R392.7

李晓蓉, 唐青海, 卜宾, 毛倩倩, 李羽, 唐存多, 姚伦广, 焦铸锦, 阚云超. 猪细胞周期蛋白依赖性激酶2多克隆抗体的制备与鉴定[J]. , 2015, 42(8): 1956-1962.

LI Xiao-rong, TANG Qing-hai, BU Bin, MAO Qian-qian, LI Yu, TANG Cun-duo, YAO Lun-guang, JIAO Zhu-jin, KAN Yun-chao. Preparation and Identification of Polyclonal Antibodies against Porcine Cyclin Dependent Kinase 2[J]. , 2015, 42(8): 1956-1962.

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猪细胞周期蛋白依赖性激酶2多克隆抗体的制备与鉴定

Preparation and Identification of Polyclonal Antibodies against Porcine Cyclin Dependent Kinase 2

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