J Vet Sci.  2007 Jun;8(2):175-180. 10.4142/jvs.2007.8.2.175.

Purification and characterization of two larval glycoproteins from the cattle tick, Boophilus annulatus

• El Hakim A
• Shahein YE
• Abouelella AM
• Selim ME

• KMID: 1089670
• DOI: http://doi.org/10.4142/jvs.2007.8.2.175

Abstract

The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.

Keyword

Boophilus annulatus; glycoproteins; larvae; vaccination

MeSH Terms

Amino Acid Sequence
Animals
Cattle
Cattle Diseases/immunology/*parasitology/prevention & control
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
Glycoproteins/immunology/*isolation & purification
Immunoblotting
Isoelectric Focusing
Ixodidae/chemistry/*immunology
Male
Molecular Weight
Rabbits
Sequence Analysis, Protein
Tick Infestations/immunology/parasitology/prevention & control/*veterinary

Figure

• 

  Fig. 1 Affinity chromatography of rabbit anti-larval antigens on a protein G-Sepharose column (1.6 × 4 cm). Three ml of antisera was applied to a column equilibrated and washed with 20 mM PB, pH 7.4, and the bound proteins were eluted with 0.1 M glycine-HCl, pH 2.5, at a flow rate of 60 ml/h.

• 

  Fig. 2 Affinity chromatography of whole larval antigens on CNBr-activated Sepharose coupled to IgG from rabbit anti-whole larval proteins (1.6 × 5 cm). The unbound proteins were washed out using 20 mM PB, pH 7.4, and the bound immunogens were eluted using 0.1 M glycine HCl buffer, pH 2.5, at a flow rate of 48 ml/h.

• 

  Fig. 3 Affinity chromatography of larval immunogens on a Con-A Sepharose column (1.6 × 4 cm). The unbound proteins were washed out using 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4, and the bound glycoproteins were eluted with 0.2 M methyl α-D glucopyrinoside at a flow rate of 30 ml/h.

• 

  Fig. 4 Electrophoretic pattern of the larval GLPs in 14% SDS-PAGE. Lane 1; molecular weight markers, Lane 2; larval GLPs, Lane 3; whole larval proteins. The gel was stained with 0.1% Coomassie blue R-250 in the fixative solution [methanol/acetic acid/water (45 : 10 : 45)] for 1 h, and was then destained by repeated soaking in the fixative.

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  Fig. 5 Analytical isoelectrofocusing (3.5-10) of larval antigens and isolated larval GLPs. Lane 1; whole larval antigens, Lane 2; larval GLPs.

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  Fig. 6 Immunoblotting of 14% SDS-PAGE. Lane 1; Con A against rabbit anti-whole larval proteins, Lane 2; larval GLPs against rabbit anti-gut proteins, Lane 3; larval GLPs against rabbit anti-whole larval proteins, Lane 4; larval GLPs against rabbit anti-whole tick proteins, Lane 5; whole larval proteins against normal rabbit serum, Lane 6; whole larval proteins against rabbit anti-whole larval proteins.

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  Fig. 7 Amino acid comparison of the N-terminal sequence of the B. annulatus larval GLPII (15 kDa) and the most significant similar sequences in the protein data bank; the integral membrane protein 2B from Rattus norvegicus (Accession number: Q5XIE8) and Gallus gallus (Accession number: O42204).

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