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. 2016 Dec;45(12):1610-1617.

Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M

Davoud Afshar  1 , Mohammad Reza Pourmand  2 , Mahmood Jeddi-Tehrani  3 , Ali Akbar Saboor Yaraghi  1 , Mohammad Azarsa  1 , Fazel Shokri  4
Affiliations

Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M

Davoud Afshar et al. Iran J Public Health. 2016 Dec.

Abstract

Background: Choline-binding proteins (CBPs) are a group of surface-exposed proteins, which play crucial and physiological roles in Streptococcus pneumoniae. The novel member of CBPs, choline-binding protein M (CbpM) may have binding activity to plasma proteins. This study aimed to clone and express CbpM and demonstrate its interaction with plasma proteins and patients' sera.

Methods: The total length of cbpM gene was cloned in pET21a vector and expressed in BL21 expression host. Verification of recombinant protein was evaluated by Western blot using anti-His tag monoclonal antibody. Binding ability of the recombinant protein to plasma proteins and the interaction with patients' sera were assessed by Western blot and ELISA methods.

Results: The cbpM gene was successfully cloned into pET21a and expressed in BL21 host. Binding activity to fibronectin and fibrinogen and antibody reaction of CbpM to patients' sera was demonstrated by Western blot and ELISA methods, respectively.

Conclusion: CbpM is one of the pneumococcal surface-exposed proteins, which mediates pneumococcal binding to fibronectin and fibrinogen proteins.

Keywords: Choline-binding protein M; Fibrinogen; Fibronectin; Streptococcus pneumoniae.

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Figures

Fig. 1:
Fig. 1:
Electrophoresis of digested recombinant vector and PCR product on agarose gel (1%). Lane 1 and 2 digested vectors with Xho-1 and Nde-1 restriction enzymes, respectively; lane 3: cbpM PCR product (387bp) and lane 4 is double digest of recombinant vector. Lane 5: DNA Marker (1Kb)
Fig. 2:
Fig. 2:
Identification and analysis of the recombinant protein by SDS-PAGE (12.5%). Lane M: Protein molecular weight marker: 14.4–116 kDa; Lane 1: whole bacteria before induction; Lane 2 to Lane 4: whole bacteria 1, 4 and 8h after induction; Lane 5: Pellet of the sonicated lysate, 6: Supernatant of the sonicated lysate
Fig. 3:
Fig. 3:
SDS-PAGE analysis of purified recombinant protein. Lane M: protein molecular weight marker14.4-116Kda, lane T0: un-induced BL21DE3, lane T4: induced BL21DE3, lane B: binding stage, lane W1: first wash, laneW5: fifth wash, lanes E1, E5 and E6 are elution stages
Fig. 4:
Fig. 4:
Western blot analysis of purified recombinant protein CbpM. Lane M: Protein size marker (Sinaclon, Iran); lane 1: Recombinant protein western blotting with His-Tag monoclonal antibody
Fig. 5:
Fig. 5:
Analysis of interaction between CbpM and fibrinogen proteins. Lane M: stained protein marker (Takapozist, Iran); Spr1754 (36KDa) and ScaA (32KDa) proteins are negative and positive controls, respectively. CbpM has positive interaction with fibrinogen
Fig. 6:
Fig. 6:
Analysis of interaction between CbpM and Fibronectin proteins. Lane M: stained protein marker (Takapozist, Iran); Spr1754 (36KDa) and ScaA (32KDa) proteins are negative and positive controls, respectively. CbpM has positive interaction with fibronectin
Fig. 7:
Fig. 7:
Anti-CbpM titers in patients’ sera with pneumococcal infection. S1toS10 represent patient’s sera. Control groups are sera obtained from healthy individuals
See this image and copyright information in PMC

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