Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (23): 4916-4923.doi: 10.3864/j.issn.0578-1752.2012.23.019

• ANIMAL SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Prokaryotic Expression of Bombyx mori Insulin-Related Peptide-Binding Protein 2 (BmIBP2) and Its Tissue-Specificity in Silkworm

 GAO  Kun, DENG  Xiang-Yuan, ZHU  Feng, WANG  Sheng-Peng, QIAN  He-Ying, GUO  Xi-Jie   

  1. 1.Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu
    2. Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, Jiangsu
    3.College of Biotechnology and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu
  • Received:2012-05-02 Online:2012-12-01 Published:2012-09-16

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Abstract: 【Objective】 The objective of this study is to express insulin-related peptide-binding protein 2 from silkworm (BmIBP2) which belongs to the immunoglobulin (Ig) superfamily in vitro and prepare polyclonal antibody against recombinant BmIBP2 for the further research about its biological function．【Method】 The gene fragment of the mature BmIBP2 peptide was amplified by PCR method with the cDNA synthesized from silkworm midgut. In order to express the protein of mature BmIBP2 peptide, the DNA segment was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. Polyclonal antibody was prepared to study the tissue-specificity expression of BmIBP2 protein in silkworm midgut. 【Result】 The SDS-PAGE result showed that the cloned recombinant BmIBP2 (rBmIBP2) was expressed in the form of inclusion bodies in E. coli BL21 with a molecular weight of 30 kD, and Western blot using the antibody against His-tag as the primary antibody indicated that rBmIBP2 had satisfied immunobiological activity. Pure protein was obtained by Ni2+ NTA resin column to prepare the anti-rBmIBP2 polyclonal antibody by immunizing New Zealand white rabbit. Western blot analysis showed that BmIBP2 protein could be bound with the polyclonal antibody in silkworm midgut with a molecular weight of 28 kD, which is consistent with the predicted molecular of BmIBP2 mature peptide．However, no specific bands were found in other tissues including fat body, hemocytes, silk gland, testicle and ovary. 【Conclusion】 Recombinant BmIBP2 protein was successfully expressed in E. coli BL21, and its polyelonal antibody was prepared in this study. Moreover, Western blot showed that BmIBP2 was correctly expressed in vitro and had a tissue-specificity expression in silkworm midgut.

Key words: Bombyx mori , insulin-related peptide-binding protein 2 (IBP2) , prokaryotic expression , purification , antibody

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