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

Abstract

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

CLC Number:

R392.7

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

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