Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus

Abstract

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven a helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.

Identification of barramundi (Lates calcarifer) and tilapia (Oreochromis spp.) fillets by DNA- and protein-analytical methods

“…9). Recently, Lee et al (2007) reported on the cloning, expression and phylogenetic analysis of parvalbumin in O. mossambicus.…”

Tropomyosin from tilapia (Oreochromis mossambicus) as an allergen

“…Parvalbumin from tilapia was described by Lee et al [30]. Parvalbumin from tilapia was described by Lee et al [30].…”

Authentication of closely related scombrid, catfish and tilapia species by PCR-based analysis and isoelectric focusing of parvalbumin

“…Different isoforms of parvalbumin have been identified in several catfish species (C. gariepinus, H. longifilis, Chrysichthys auratus) [47], tilapia [76] and snappers (Lutjanus argentimaculatus, L. johnii) [55]. Recently, the expression and phylogenetics of parvalbumin in tilapia (O. mossambicus) have been studied by Lee et al [54].…”

“…Information on this allergen has been recently reported for tuna [49][50][51][52][53], but knowledge of parvalbumin genes and their expression in tilapia and snapper is limited [54][55][56]. IEF of sarcoplasmic proteins has been applied to identify P. hypophthalmus, P. bocourti [57] and other catfish species [58].…”