Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus.
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 alpha 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.
Lee
Shyh-Jye
SJ
Institute of Zoology, National Taiwan University, Taipei, Taiwan 106, R.O.C. jefflee@ntu.edu.tw
Ju
Chi-Ching
CC
Chu
Shian-Ling
SL
Chien
Ming-Shan
MS
Chan
Tun-Hao
TH
Liao
Wen-Liang
WL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D015870:Gene Expression; D017403:In Situ Hybridization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D017433:Protein Structure, Secondary / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D017210:Tilapia / Q000235:genetics*
D017931:DNA Primers; D010320:Parvalbumins