Cloning of Qiantang River triangular bream (Megalobrama terminalis) IGF-I gene and expression of the recombinant pre-IGF-I in Escherichia coli.
The insulin-like growth factor (IGF-I) gene (GenBank accession no. AY247412) of Qiantang River triangular bream (Megalobrama terminalis) was cloned for the first time from the liver by reverse transcriptase polymerase chain reaction. The gene was inserted into pMD 18-T vector to construct the recombinant plasmid pMD 18-T/IGF-I. Sequence analysis indicated that the IGF-I cDNA consisted of 486 nucleotides encoding 161 amino acids, which spanned the complete signal peptide, mature peptide (including B, C, A, and D domains), and E-domain. Analysis of the E domain indicated that triangular bream IGF-I gene belonged to the IGF-I Ea-2 subtype. To construct the expression plasmid, the IGF-I gene was subcloned into prokaryotic expressing vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IGF-I was transformed into Escherichia coli BL21 (DE3), and the transgene expression was observed after being induced with isopropylthiogalactoside. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting indicated that the recombinant fusion protein had immune activity, and the molecular weight was about 47 kDa. The results of SDS-PAGE and thin-layer scanning showed that the yield of fusion protein had been enlarged with prolonging time. When the time of induced expression was 1, 2, 3, 4, 5, and 6 h, the expression amount was approximately 1.4, 4.3, 8.1, 11.3, 16.3, and 18.8% of total bacterial protein, respectively.
Zhang
Haihua
H
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, People's Republic of China.
Tong
Fu-Dan
FD
Lu
Qing-Er
QE
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005796:Genes; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D045483:Rivers; D014871:Water Microbiology
D007334:Insulin-Like Growth Factor I