Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus (Boophilus) annulatus salivary gland.
Immunoscreening of a cDNA expression library of the Rhipicephalus (Boophilus) annulatus tick with purified rabbit anti-R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661bp sequence. The sequence includes an open reading frame of 543bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C.
Shahein
Yasser E
YE
Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, El-Tahirir St., Dokki, Cairo 33211, Egypt. yassershahein_nrc@yahoo.com
El-Rahim
Mohamed T Abd
MT
Hussein
Nahla A
NA
Hamed
Ragaa R
RR
El-Hakim
Amr E
AE
Barakat
Maged M
MM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D050888:Heat-Shock Proteins, Small / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015151:Immunoblotting; D008969:Molecular Sequence Data; D055550:Protein Stability; D048494:Rhipicephalus / Q000235:genetics*; D012469:Salivary Glands / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013696:Temperature; D017186:Titrimetry
D000906:Antibodies; D018076:DNA, Complementary; D050888:Heat-Shock Proteins, Small