Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (1): 181-190.doi: 10.3864/j.issn.0578-1752.2019.01.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm (Bombyx mori)

ZHANG Kui,LI ChongYang,SU JingJing,TAN Juan,XU Man,CUI HongJuan()   

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
  • Received:2018-08-08 Accepted:2018-08-24 Online:2019-01-01 Published:2019-01-12
  • Contact: HongJuan CUI E-mail:Hongjuan.cui@gmail.com

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Abstract:

【Objective】The objective of this study is to analyze the gene sequence and structural characteristics of integrin β2 in silkworm (Bombyx mori), and its expression profile in hemocytes following the larval exposure to different bacterial pathogens, investigate the binding and agglutination properties of the recombinant integrin β2 protein to various pathogen-associated molecular patterns (PAMPs) and bacteria, which will lay a foundation for further exploring the protein function of integrin β2 in B. mori. 【Method】Bioinformatics tools were used to determine the sequence and structural characteristics of integrin β2, and real-time fluorescent quantitative PCR (RT-qPCR) assay was executed to evaluate expression profile in hemocytes after microbial (Escherichia coli and Staphylococcus aureus) challenge. The cDNA fragment of integrin β2 was amplified using PCR, and the fragment containing the extracellular domain was inserted into a prokaryotic expression vector (pET22b). The insertion was confirmed in the recombinant plasmid and transformed into E. coli Rosetta (DE3), and then induced by IPTG to produce recombinant protein. The recombinant protein was purified using Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western blot. ELISA and Western blot were executed to determine the binding abilities of the recombinant protein to PAMPs (LPS and PGN) and different bacteria. Moreover, the agglutination ability and bacterial clearance assay were performed to understand the specific biological roles of integrin β2 in immunity.【Result】B. mori integrin β2 contains typical integrin β subunits, which comprises a long extracellular domain, a single transmembrane region and a short cytoplasmic tail. Further, it has several conserved motifs such as the MIDAS, EGF domain, Cys-repeat sequences and NPxY motifs. The RT-qPCR analysis showed that the integrin β2 expression varied significantly in hemocytes after infection with bacteria. High purity recombinant protein was obtained by prokaryotic expression and protein purification. The results of SDS-PAGE and Western blot showed that the purified recombinant protein was of high purity and could be used in subsequent tests. ELISA assay indicated that the purified recombinant integrin β2 protein had a strong binding ability to PAMPs (LPS and PGN). The results of bacterial binding test showed that the recombinant protein could bind many bacteria, but the binding ability with Gram-positive bacteria was higher than that with Gram-negative bacteria. The agglutination assay showed that the recombinant protein had strong agglutination effects on S. aureus in the presence of ca 2+. Further, bacterial clearance assay suggested that the recombinant protein could effectively promote the cleaning of exogenous invading bacteria from B. mori. 【Conclusion】The integrin β2 has a typical structure of the β integrin family, and it can recognize PAMPs (LPS and PGN) and enhance the aggregation of invading microbial pathogens by directly binding to them. Taken together, integrin β2 may play an important biological role in the bacterial immune response of B. mori.

Key words: Bombyx mori, integrin β2;, microbial challenge, prokaryotic expression, bacterial agglutination

Fig. 1

Protein sequence alignment of integrin β"

Fig. 2

Expression analysis of integrin β2 after induced by microbes *P<0.05 vs control PBS, **P<0.01 vs control PBS, Student’s t-test (n=3)"

Fig. 3

The prokaryotic expression and protein purification of integrin β2"

Fig. 4

Binding of the recombinant integrin β2 protein to PAMPs BSA used as the control"

Fig. 5

Western blot analysis of the binding of the recombinant integrin β2 protein to bacteria"

Fig. 6

Microorganism agglutination activities of the recombinant integrin β2 protein to S. aureus"

Fig. 7

In vivo bacterial clearance activities of rIntegrin β2"

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