Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.
Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.
Hu
Xi-Lian
XL
College of Life Sciences, Zhejiang University, Hangzhou, China.
Zhu
Mu-Yuan
MY
Zhang
Zhi-He
ZH
Hou
Rong
R
Shen
Fu-Jun
FJ
Li
Fu-Zhen
FZ
Zhang
An-Ju
AJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D046010:Tigers / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I