Molecular cloning, expression and functional characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene from mandarin fish (Siniperca chuatsi).
Interferon-γ-inducible lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. For this important function in the immune system, we cloned a GILT gene homologue from mandarin fish (designated mGILT), a kind of precious freshwater fish with high market value. Through reverse transcription PCR and rapid amplification of cDNA ends (RACE) strategies, we obtained the full-length cDNA of mGILT, which consists of 1008 bp with a 771 bp open reading frame, encoding a protein of 256 amino acids, with a putative molecular weight of 28.47 kDa. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 6 conserved cysteines. The result of real-time quantitative PCR showed that mGILT mRNA was expressed in a tissue-specific manner. In addition, the expression of mGILT mRNA was obviously up-regulated in splenocytes and kidney after induction with lipopolysaccharide (LPS). Recombinant mGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Further study revealed that mGILT exhibit thiol reductase activity on IgG substrate. These results suggest mGILT is highly likely to play a role in the immune responses in mandarin fish.
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Jinyun
J
Song
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Hongzhen
H
Liu
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Lei
L
Ma
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Li
L
Ma
Medical Department, Yancheng Institute of Health Sciences, Yancheng 224005, China.
Cuixiang
C
Gao
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Jiangsu Province Key Laboratory for Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Medical Department, Yancheng Institute of Health Sciences, Yancheng 224005, China. Electronic address: shuangquanz@yahoo.com.
Shuangquan
S
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008070:Lipopolysaccharides; D008969:Molecular Sequence Data; D009928:Organ Specificity; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics* / Q000378:metabolism*; D010473:Perciformes / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment / Q000662:veterinary; D029961:Zebrafish Proteins
D029941:Fish Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C578853:ifi30 protein, zebrafish
Interferon-γ-inducible lysosomal thiol reductase; Mandarin fish; Real-time quantitative PCR; Thiol reductase activity; cDNA cloning