Construction expression and purification of C reactive protein

Zhi-Nong YIN; Xin ZHOU; Fang ZHENG; Lei ZHAO; Yong XIA; Han-Ning HU; Xin-Qiang JIANG

Objective To build a foundation for determination of C reaction protein,C reaction protein was expressed and purified,and the immune reactivity of the purified protein was identified.Methods The CRP cDNA was amplified by RT-PCR from human liver cDNA library and inserted into expression vector pCRTT/NT.The recombined plasmid CRP-pCRTT/NT which expressed the fusion protein of CRP was then transferred into lysogenic host strain E coli.BL21 (DE3).The target protein was identified using SDS- polyacrylamide gel electrophoresis (SDS-PAGE).Affinity chromatography was used for protein purification.The immune reactivity of purified CRP was identified by Western blot using anti-CRP specific antibody.Results Recombiant human CRP was expressed in inclusion bodies of E.coli with a six histamine tag.The purify of recombinant protein was detected by SDS-PAGE as a single band at 30 000 and was identified by Western blot.Conclusions A plasmid expressed CRP protein is constructed and the purification system of CRP protein is established.The immune reactivity of the purified protein is identified by Western blot,which makes a good base for the preparation of CRP test kit.

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Construction expression and purification of C reactive protein / 中华检验医学杂志