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Cloning of Qiantang River Triangular Bream (Megalobrama terminalis) IGF-I Gene and Expression of the Recombinant pre-IGF-I in Escherichia coli

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Abstract

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.

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Acknowledgements

This project was supported by Zhejiang Provincial Natural Science Foundation of China (No. 300265).

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Authors and Affiliations

  1. College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, People’s Republic of China

    Haihua Zhang & Fu-Dan Tong

  2. College of Animal Science, Zhejiang University, Hangzhou, 310029, People’s Republic of China

    Haihua Zhang & Fu-Dan Tong

  3. Hangzhou Academy of Agriculture Science Research, Hangzhou, 310024, People’s Republic of China

    Qing-Er Lu

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  1. Haihua Zhang
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  2. Fu-Dan Tong
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  3. Qing-Er Lu
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Correspondence to Fu-Dan Tong.

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Zhang, H., Tong, FD. & Lu, QE. Cloning of Qiantang River Triangular Bream (Megalobrama terminalis) IGF-I Gene and Expression of the Recombinant pre-IGF-I in Escherichia coli . Appl Biochem Biotechnol 150, 15–24 (2008). https://doi.org/10.1007/s12010-007-8027-9

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  • DOI: https://doi.org/10.1007/s12010-007-8027-9

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