Journal of Jilin University Medicine Edition ›› 2015, Vol. 41 ›› Issue (04): 864-869.doi: 10.13481/j.1671-587x.20150438

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Preparation of polyclonal antibody against non-structural NP1 of minute virus of canine and its property identification

YAN Yan, ZHANG Qian, MA Jing, LI Jianning, ZHANG Wei, SUN Yuning   

  1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China
  • Received:2014-11-19 Published:2015-08-01

Abstract:

Objective To prepare the polyclonal antibody against non-structural NP1 of minute virus of canine (MVC), and to identify its properties. Methods The target NP1 gene of MVC was amplified from MVC infection clone (pI-MVC),and was inserted into the pGEX-4T-3 vector to form the recombinant plasmid. Then the prokaryotic expression vector of pGEX-4T-3-NP1 was transform into E.coli (BL21),and the GST-NP1 fusion protein was induced under the optimized induction of isopropyl β-D-1-thiogalactopyranoside(IPTG).The recombinant proteins were purified using affinity chromatography.The purified fusion protein was inoculated into the adult rabbits to develop antiserum.After the titer of the antiserum was detected by ELISA,Western blotting and immunofluorescence methods were performed to evaluate the features of the prepared antiserum. Results The prokaryotic expression vector pGEX-4T-3-NP1 of MVC NP1 gene was successfully constructed.The soluble recombinant protein was highly expressed in E.coli BL21,and then it was purified and inoculated into the adult rabbits to obtain high titer antiserum.The ELISA result showed that the titer of the antiserum was 1:400 000.The Western blotting and immunofluorescence results showed that the specificity of the prepared antiserum was perfect. Conclusion The antiserum of NP1 prepared from MVC GST-NP1 fusion protein has shown a high titer and high specificity against NP1 proteins,which can be used for further study of NP1 in the molecular mechanisms of MVC infection.

Key words: minute virus of canine, NP1 gene, polyclonal antibody

CLC Number: 

  • Q78