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| The Soluble Expression and Antibacterial Activity of C Terminus Region Lysozyme Gene(SjLys-C) from Sea Cucumber(Stichopus Japonicus) |
| chang yi hai1,Cong Li na2,LU Dong 1,wang hong ying1 |
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2. Dalian Polytechnic University
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Abstract In order to characterize the antibacterial activity of gene products expressed by several gene fragments from the gene of Stichopus japonicus lysozyme (SjLys), its cDNA (GenBank accession No. EF036468) was analysed by bioinformatics. The result showed that the amino acid region of C terminus of SjLys contained non-enzymatic activity. Therefore, a pair of primers including NcoⅠ and EcoRⅠrestriction sites were designed according to the cDNA sequence of SjLys and the gene fragment of C terminus of SjLys (SjLys-C) was amplified by RT-PCR from the total RNA of the S. japonicus intestine. As a result, the target gene was obtained at a length of 259 bp fragment. The fragment of SjLys-C was subcloned into the expression vector of pET-32a(+) to construct the recombinant plasmid of pET-32a(+)-SjLys-C. Then the recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS to gain the genetically engineering strain pET-32a(+)-SjLys-C/ E. coli Rosetta(DE3)pLysS. We used the strain to induce and express the recombinant protein SjLys-C. The result showed that the genetically engineering strain could highly express the recombinant protein of 26 kD. Moreover, the recombinant protein SjLys-C could express in solube form, which was taken up 70% of the total protein. Western blotting analysis found that the recombinant protein SjLys-C had a specific immune response with Penta-His antibody at the position of about 26 kD. Therefore, it is evidence that the recombinant protein SjLys-C must be the target protein. The antibacterial activity of the purified recombinant protein SjLys-C was also analyzed. The recombinant protein SjLys-C displayed inhibitive effect on the growth of the Micrococcus lysodeikticus and Vibrio parahaemolyticus. In particular, the heat-treated recombinant protein SjLys-C inhibitive activities were enhanced from5% to 21% at 100℃and 40 min treatment. These results showed that the strain pET-32a(+)-SjLys-C/E. coli Rosetta(DE3)pLysS could produce recombinant protein SjLys-C in a soluble form and exhibit the potent antibacterial activity. Therefore, the product will be widely used in agriculture and medicine and has great market potential and development value.
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Received: 28 November 2011
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Corresponding Authors:
Cong Li na
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